L-amino acid oxidase with cytotoxic activity from aplysia functata

ABSTRACT

The present invention relates to a cytotoxic polypeptide which is an L-amino acid oxidase isolated from the ink of the sea hare  Aplysia punctata.

This application is a divisional application of U.S. application Ser. No. 10/542,769, filed on Mar. 6, 2006, which was the National Stage application under §371 of PCT/EP04/00423, filed Jan. 20, 2004.

DESCRIPTION

The present invention relates to a cytotoxic polypeptide which is an L-amino acid oxidase isolated from the ink of the sea hare Aplysia punctata.

The sea hare Aplysia produces a pink-coloured ink, which has cytotoxic activity towards several eukaryotic cell lines. WO97/16457 discloses a partial sequence from an Aplysia protein, which allegedly has anti-tumor activity. Cyplasin L (558 aa, NCBI accession number 11967690) and cyplasin S (421 aa, 11967688; Petzelt and Werner, 2001, Cell Biology International, 25(2):A23) both include parts of sequences disclosed in WO 97/16457. Cyplasin S exhibits 95% sequence identity to cyplasin L. Cyplasin L is produced in the nidamental gland but neither in the ink gland (including the mantle region) nor in the opaline gland of Aplysia punctata. Thus, it is concluded that cyplasin is not a component of Aplysia ink and is not responsible for the cytotoxic activity of the Aplysia ink. A detailed description of Aplysia anatomy and a dissection guide can be found in the internet in Richard Fox, Invertebrate anatomy (1994, available via the world wide web at science.lander.edu/rsfox/).

The overall aim in tumor therapy is the selective eradication of transformed cells without harming healthy cells. Several glycoproteins isolated from sea hares (Aplysia species) have attracted attention because of their anti-tumor activity, e.g. aplysianin A from Aplysia kurodai, or cyplasins. The underlying mechanism for such activity has however not been elucidated so far. Recombinant intracellular cyplasins seem to be non-toxic, whereas the extracellular cyplasin is cytotoxic (Petzelt et al., Neoplasia, 4:49-59, 2002).

WO 03/057726 discloses a cyplasin which is devoid of a functional secretory signal sequence. Since cyplasin only causes eukaryotic cell death from outside, the cyplasin of WO 03/057726 can thus be functionally expressed in eukaryotic cells without killing these cells. When acting from outside, cyplasin induced cell death is accompanied by fast depolymerization of the actin filaments. Expression of bioactive cyplasin S and L in prokaryotic host cells is not possible.

WO 02/31144 discloses a further cytotoxic factor isolated from the ink of Aplysia punctata. Fragments of the amino acid sequence of the factor are disclosed. No data were presented demonstrating that this factor has any oxidase function or has any properties related to an oxidase.

At least two main phenotypes of cell death are described: apoptosis, a genetically fixed physiological form of cell death, is accompanied by shrinkage, membrane blebbing, nuclear fragmentation, and final disintegration into so-called apoptotic bodies. In contrast, necrosis is a pathological process characterized by membrane disruption and cell swelling. Cell death induced by reactive oxygen and nitrogen species (ROS/NOS) might lead to apoptosis and necrosis but also to other forms of cell death, which cannot be clearly assigned to one of these main forms of cell death.

The cytotoxic factors derived from the sea hares so far have several disadvantages which might hamper its application. The biological function and the nature of the cytotoxic activity, which are prerequisites for the development of a lead compound, are not known so far. Aplysianin A contains a dinucleotide binding fold and the so-called “GG motif” which are found in many flavoproteins. The GG motif has also been described in cyplasins (Petzelt et al., supra). Based on this knowledge, the factors can be applied in its entirety only, because the domains relevant for proper function and cellular receptors are unknown. The administration of an entire non-self protein to an animal or a human might cause severe immunologic complications.

The dinucleotide binding fold and the GG motif are found e.g. within the N-terminal domain of FAD containing enzymes (e.g. reductases, dehydrogenases, hydroxylases, peroxidases, and oxidases). FAD containing enzymes can be classified into five groups GR1, GR2, FR, PCMH, and PO according to the sequences of their FAD binding domains and additional conserved sequence motifs (Dym and Eisenberg, Protein Science, 10:1712-1728, 2001). The consensus sequence of GR1 and GR2 is GxGxxG. The GG motif RhGGRhxxT/S (SEQ ID NO: 76) is commonly found in oxidases, e.g. L-amino acid oxidases, monoamino oxidases, polyamine oxidases, and putrescine oxidases, wherein x describes any amino acid, and h describes a hydrophobic amino acid.

L-amino acid oxidases catalyse the formation of H₂O₂, ammonia, and an alpha keto acid from an amino acid in the presence of oxygen and H₂O (Geyer et al, 2001, Eur. J. Biochem. 268, 4044-4053). An L-lysine alpha oxidase (EC 1.4.3.14) for instance can be obtained from the fungus Trichoderma spec. (Kusakabe et al., J. Biol. Chem. 10:976-981, 1980) which shows antimetastatic effects (Umanskii et al., Biull Eksp Biol Med. 109:458-9, 1990, Khaduev et al., Biull Eksp Biol Med. 112:419-22, 1991). The Trichoderma L-lysine oxidase is a dimer with a molecular weight of 112-119 kDa. A further L-lysine oxidase obtained from the fish Chub mackerel is a dimer and has a molecular weight of 135 kDa Jung et al., J. Immunol. 165:1491-1497, 2000) and induces apoptosis. Apoxin is an L-leucin oxidase from the rattlesnake (Crotalus atrox) venom which induces apoptosis in tumor cells and vascular endothelial cells in vitro (Torii et al., J. Biol. Chem. 272:9539-9542, 1997). A cytotoxic L-lysine alpha oxidase is described in the art which penetrates into Jurkat cells and there activates oxidative deamination of L-lysine and correspondingly the peroxide formation. Conjugates of the enzyme with monoclonal antibodies against the CD5 receptor cannot penetrate into the cells and are assumed to produce toxic H₂O₂ outside the cells. The conjugates have a reduced cytotoxic effect, although the effect of conjugation upon enzymatic activity is negligible (Zhukova et al., Vopr Med Khim 2001, 47:588-592). Another L-lysine oxidase obtained from the snail Achatina fulica and producing H₂O₂ is found to have an antimicrobial effect. This oxidase might be useful as an agent against pathogenic bacteria (Ehare et al., 2002, FEBS Letters, 531:509-512).

Most known alpha amino acid oxidases which produce H₂O₂ possess a broad substrate specificity. The L-lysine alpha oxidase from Trichoderma viride (EC 1.4.3.14, Kusakabe et al., supra) is specific for lysine, but also oxidizes L-ornithine, L-phenylalanine, L-tyrosine, L-arginine, and L-histidine to a lesser extent. The L-lysine oxidase of Chub mackerel (EMBL, AJ400781; Jung et al., supra) is specific for lysine and in addition transforms arginine, histidin, leucine, methionine, phenylalanine, and ornithine (specificity 40 fold reduced). Even if these enzymes could be cytotoxic due to their ability to produce H₂O₂, a therapeutic use is hampered because substrates of these enzymes are available in the body fluid in amounts sufficient to release H₂O₂ everywhere in the body. Under these conditions, possible negative side effects of H₂O₂ are difficult to eliminate.

In addition to H₂O₂ producing enzymes, cells possess a detoxification system which eliminates reactive oxygen species (ROS), in particular H₂O₂. An important class of detoxifying peroxidases are peroxiredoxins. Peroxiredoxins comprise a class of highly conserved oxidases. In mammals, six different isoforms are known which catalyze the reduction of peroxides by using reducing equivalents that are provided by thioredoxin or glutathone. During catalysis, peroxiredoxin I (Prx I) is inactivated by oxidation of the active site cysteine to cysteine sulfinic acid, a modification which is reversible upon removal of H₂O₂. Previously, overexpression of both Prx I and Prx II has been shown to render cells resistant to H₂O₂ induced apoptosis.

The problem underlying the present invention is the provision of a means for selective generation of H₂O₂ in target tissues, e.g. in tumor tissues with less toxic side effects upon normal cells. The solution is a cytotoxic polypeptide which can be isolated from the ink of the sea hare Aplysia punctata and which is a specific L-lysine and/or L-arginine oxidase producing H₂O₂ or a fragment or derivative of said polypeptide. The activity of the enzyme can be modulated be administration of substrate. The enzyme provides a lead structure, and it can be used for target identification.

A first aspect of the present invention is a purified polypeptide which exhibits cytotoxic activity on tumor cells and which comprises the amino acid sequence shown in SEQ ID NO: 2, 4, or 6, or a cytotoxic fragment thereof. These sequences are derived from a cytotoxic 60 kDa protein purified from crude ink of Aplysia punctata via anion exchange chromatography and gel filtration (see examples 1 and 4). Thus, the polypeptide or the fragment is termed APIT (Aplysia punctata ink toxin). The purity of the fractions can be determined by SDS-PAGE and silver staining.

The cytotoxic activity of APIT or the diluted crude ink can be measured by the reduction of the metabolic activity of eukaryotic cells. A person skilled in the art knows suitable methods and cell lines. For example, the metabolic activity of Jurkat T cells can be measured by the addition of WST-1, which is a tetrazolium salt converted by cellular enzymes of viable cells, e.g. by the mitochondrial dehydrogenase, to a dark red formazan. Therefore, the amount of formazan correlates with cell vitality. Formazan can be determined photometrically at 450 nm. Further, dead eukaryotic cells killed by APIT or the diluted crude ink can be counted by adding propidium iodide (PI) at 1 μg/ml in PBS and subsequent flow cytometer analysis. PI is a DNA binding dye which is taken up by dead cells with permeable membranes.

The cytotoxic activity of APIT is reduced by at least 70% after 10 min incubation at 60° C. At 70° C., the activity is almost absent, whereas 0° C. to 50° C. have no effect upon the activity. APIT shows a loss of activity with decrease of pH, with complete inactivation after 10 min pre-incubation at pH 3. After 30 min treatment with 6 M urea, the activity of APIT is almost unaffected. At 8M urea, the activity is reduced by about 50% (example 3).

Tumor cells treated with APIT displays a morphology which is neither typical for apoptosis nor for necrosis but rather is typical for oxidative damage induced cell death. Shrunken nuclei and lack of cell swelling are apoptotic, and early membrane permeabilization is a necrotic characteristic (example 2). The phenotype induced by APIT could be reproduced in Jurkat cells by treatment of the cells with concentrations of H₂O₂>200 μM, indicating that H₂O₂ is the active compound in APIT cytotoxic effect. H₂O₂ concentrations <100 μM induced apoptosis in Jurkat cells. In contrast to the mode of action of cyplasins, a depolymerization of the active filaments cannot be observed in APIT induced cell death, indicating that the mechanism of APIT action is distinct from that of cyplasins (Example 12).

By depriving possible substrates which can be converted into H₂O₂ from the culture medium of the tumor cells, it can be demonstrated that no further toxic effect of APIT upon tumor cells is present. Deprivation of L-Iysine and L-arginine from the medium prevents cell death completely. This phenomenon can be observed within a period of 6 to 8 hours during cultivation of tumor cells. In a detailed analysis of the enzymatic activity of APIT, media containing single amino acids (20 L-amino acids, D-lysine) confirmed that L-lysine and/or L-arginine is converted into H₂O₂ and the respective alpha keto acid to the same extent, whereas no conversion could be measured with any other of the remaining 18 L-amino acids and D-lysine (example 7). The production of H₂O₂ is independent of the presence of cells, however, the presence of cells reduces the amount of free H₂O₂, which might be due to detoxification of the medium by the cells. Catalase (a H₂O₂ hydrolyzing enzyme) prevents tumor cell death induced by purified APIT and by crude ink as well, confirming the conclusion that H₂O₂ is responsible for the ink mediated killing of tumor cells (example 6). Anti-tumor activity also appears after long-term in vitro treatment (>18 hours) of tumor cells by the cytotoxic factor isolated from the ink of Aplysia punctata in combination with an H₂O₂ consuming factor, like catalase. In comparison to tumor treatment with the cytotoxic factor from Aplysia punctata alone, this alternative tumor treatment takes a much longer time to become effective. The interplay of both enzyme activities continuously reduces L-lysine and L-arginine in the medium which are essential for the living of tumor cells. The tumor cells die as a result.

In summary, the data demonstrate that the polypeptide of SEQ ID NO: 2,4, or 6 (APIT) is an oxidase which is capable to produce H₂O₂. Particularly, the polypeptide is an alpha amino acid oxidase. More particularly, the polypeptide specifically converts L-lysine and/or L-arginine in the presence of O₂ and H₂O into an alpha keto acid, ammonia, and H₂O₂. Thus, the polypeptide is preferably an L-lysine and/or L-arginine oxidase.

A characteristic feature of the active fractions containing APIT purified from crude ink were two absorption maxima at 390 nm and 470 nm, a hallmark of flavoproteins. A flavine nucleoside, particularly FAD is required as a co-factor for the anti-tumor and oxidase activity of APIT as removal of FAD inactivated APIT (example 5).

Analysis of the sequences SEQ ID NO: 2, 4, and 6 revealed that APIT comprises a sequence similar to known dinucleotide binding folds which are characteristic for flavoproteins (FIG. 4 c). The GG-motif (consensus sequence RhGGRhxT/S) (SEQ ID NO: 76) is found adjacent to the dinucleotide binding fold.

A further aspect of the present invention is a polypeptide comprising a fragment of the polypeptides of the sequences of SEQ ID NO: 2, 4, or 6 which can be used as a lead structure for drug development. APIT can be digested by a protease without loss of activity. Digestion leaves the substrate specificity unaltered. Thus, the fragment exhibiting cytotoxic activity is an L-lysine and/or L-arginine oxidase. Preferably, proteinase K is used which is a relative unspecific protease resulting in small fragments. Other proteases which can be selected among specific or unspecific proteases known by a person skilled in the art can be used instead of proteinase K. The cytotoxic proteinase resistant domain of APIT is of particular importance for the development of a non-immunogenic, fully active small compound.

Further preferred fragments comprise partial amino acid sequences of APIT which are obtained by peptide mass fingerprinting, ESI/MS, and Edman degradation:

DG(I/V)CRNRRQ, (SEQ ID NO: 46) DSGLDIAVFEYSDR, (SEQ ID NO: 47) VFEYSDR, (SEQ ID NO: 48) LFXYQLPNTPDVNLEI (SEQ ID NO: 49) (X = T in SEQ ID NO: 2, 4 and 6), VISELGLTPK, (SEQ ID NO: 50) GDVPYDLSPEEK, (SEQ ID NO: 39) VILAXPVYALN (SEQ ID NO: 51) (X = M in SEQ ID NO: 2, 4 and 6), ATQAYAAVRPIPASK, (SEQ ID NO: 37) VFMTFDQP, (SEQ ID NO: 52) SDALFFQMYD (SEQ ID NO: 53) (FFQ is FSQ in SEQ ID NO: 2, 4 and 6), SEASGDYILIASYADGLK, (SEQ ID NO: 54) NQGEDIPGSDPQYNQVTEPLK (SEQ ID NO: 55) (PQY is PGY in SEQ ID NO: 2, 4 and 6)

While not wishing to be bound by theory, the FAD group which is tightly bound to the amino acid chain, e.g. by a covalent bond, might cover possible protease cleavage sites. Thus, protease treatment results in a fragment comprising the active centre of the enzyme, including the prosthetic group FAD. This conclusion is confirmed by the finding that native APIT cannot be cleaved by trypsin, but trypsin can digest denaturated APIT.

Thus, an especially preferred fragment of APIT which is an oxidase exhibiting cytotoxic activity is a sequence comprising the dinucleotide binding fold and the GG motif corresponding to amino acid residues No. 39 to 77 in SEQ ID NO: 2. This sequence is identical to the sequence of amino acid residues No. 38 to 76 in SEQ ID NO: 4 and No. 21 to 59 in SEQ ID NO: 6. More preferably, the fragment has an L-lysine and/or an L-arginine oxidase activity. Further, the fragment can comprise a stretch of additional amino acid residues which may be selected from SEQ ID NO: 2 or 4 from the sequences adjacent to the residues No. 39 to 77 in SEQ ID NO: 2 or No. 38 to 76 in SEQ ID NO: 4. Preferably, 1-20 additional amino acid can be present at the N-terminus and/or the C-terminus. More preferably, 1-10 additional amino acid can be present at the N-terminus and/or the C-terminus. Most preferably, 1-5 additional amino acid can be present. A further aspect are polypeptides which are homologous to the polypeptides of SEQ ID NO: 2,4, or 6, or to fragments thereof, which have an identity of at least 70%, preferably at least 80%, more preferably at least 90%, or most preferably at least 95%. SEQ ID NO: 2, 4, or 6 describe natural variations of APIT by replacements of single amino acids not affecting its function. In further 11 clones, four mutations were found within the sequence comprising the dinucleotide binding fold and the GG motif (Pos. 39 to 77 in SEQ ID NO: 2, see example 4). Taking into account that a fragment obtained by proteolytic digestion is still active as a L-lysine and/or L-arginine oxidase, it can be expected that further modifications of the sequence, e.g. by amino acid substitutions, deletions and/or insertions will not substantially affect the function of APIT. A modified sequence exhibits an identity of preferably at least 70%, more preferably at least 80% and most preferably at least 90% to a reference sequence, e.g. SEQ ID NO: 2. Preferably, the sequence of Pos. 39 to 77 in SEQ ID NO: 2 has a higher degree of identity to the reference sequence than the total amino acid sequence, e.g. preferably at least 33 of 39 amino acid residues (at least about 85%), more preferably 35 of 39 residues (at least about 90%), and most preferably 37 of 39 residues (at least about 95%).

A still further aspect is a polypeptide of the present invention as described above which is a recombinant polypeptide. The recombinant polypeptide is characterized as being manufactured in a heterologous, i.e. non-Aplysia host cell, e.g. in a bacterial cell such as E. coli or Bacillus, in a yeast cell such as saccharomyces cerevisiae, in an insect cell or in a mammalian cell. The recombinant polypeptide has preferably an oxidase, or, more preferably, an L-lysine and/or an L-arginine oxidase activity. Expression of the polypeptide can be done by standard expression systems known by a person skilled in the art. For proper enzymatic function, the prosthetic group FAD may have to be introduced into the polypeptide.

The protein of the invention or a fragment thereof may be in the form of a fusion protein, i.e. fused to heterologous peptide or polypeptide sequences. Preferably fusion proteins are genetic fusions, wherein the nucleic acid sequence encoding a protein or a protein fragment as described above is fused to a nucleic acid sequence encoding a heterologous peptide or polypeptide sequence. The heterologous peptide or polypeptide sequence may be selected from signal sequences, which provide desired processing and/or transport in a host cell. The signal sequence is preferably located at the N- and/or C-terminus of the APIT sequence. Further examples of heterologous sequences are domains which assist expression in host cells and/or purification from cellular extracts or culture media. Still further examples of heterologous sequences are targeting sequences which may direct the APIT polypeptide to a desired target site, e.g. in an organism. Suitable targeting sequences may be e.g. single chain antibodies, which may be directed against tumor specific antigens or proteinaceous ligand sequences, which may be directed against tumor specific receptors.

A further aspect of the present invention is a nucleic acid coding for the polypeptide as described above. The total mRNA of the mantle gland, the nidamental gland, the digestive gland, and the opaline gland can be prepared by standard methods. The mRNA can be reverse transcribed using the tagged oligo dT oligonucleotide (Oligo 1, FIG. 4 b). The tag is a random sequence not expected to be present within Aplysia mRNA to be reverse transcribed. PCR can be performed using the degenerated primer (Oligo 2) derived from the APIT peptide VFEYSDR (SEQ ID NO: 48) and the specific primer (Oligo 3) directed against the tag sequence of the oligo dT primer Oligo 1. The amplified sequence can be cloned into a standard vector and can be sequenced by standard techniques. By this strategy, the 3′ terminal sequence of the APIT gene can be obtained. The 5′ terminal sequence can be obtained by the RACE strategy. The mRNA from selected tissues (see above) is reverse transcribed using an oligonucleotide derived from the known 3′ terminal sequence (e.g. Oligo 4, or Oligo 6) and can be treated with a terminal transferase in the presence of CTP, resulting in a 3′-poly-C-sequence (at the minus strand). PCR can be performed using a tagged primer against the poly-C-sequence (Oligo 5) and a specific primer, e.g. Oligo 4, or Oligo 6. The amplified product can be cloned and sequenced by standard techniques. Finally, for obtaining full-length cDNA clones, specific primers, e.g. Oligo 8 and Oligo 9 can be used. By this strategy, three different clones were obtained and sequenced. The nucleotide sequences are described in SEQ. ID. No. 1, 3, and 5 which are identical to 97% (1560 of 1608) of the nucleotides. 42 of 48 mutations are silent mutations which have no effect upon the amino acid sequence.

By this strategy, further clones of APIT can be obtained which might have a differing sequence. Since more than ten sequences of APIT are known, specific or degenerated primers may be selected from these sequences, and new clones can be obtained by a single PCR of reverse transcribed mRNA.

Thus, the nucleic acid encoding a polypeptide as specified above preferably comprises

-   -   (a) a nucleotide sequence as shown in SEQ ID NO: 1,3, or 5, or         at least the polypeptide coding portion thereof, or the         complement thereof, or     -   (b) a nucleotide sequence corresponding to the sequence of (a)         within the scope of degeneracy of the genetic code, or the         complement thereof, or     -   (c) a nucleotide sequence hybridizing under stringent condition         with the sequence of (a) and/or (b), or     -   (d) a nucleotide sequence which is homologous to the sequences         of (a) and/or (b).

The nucleic acid may be a single stranded or double stranded nucleic acid (DNA or RNA). The nucleic acid is obtainable from natural sources e.g. from Aplysia by extraction of RNA, construction of cDNA libraries and screening of the library using degenerated oligonucleotides which were deduced from the peptide sequences described above. The nucleic acid is further obtainable by RT-PCR using RNA extracted from Aplysia and oligo-dT-primers or degenerated primers. On the other hand, the nucleic acid is obtainable by chemical synthesis.

Hybridization under stringent conditions preferably means that after washing for 1 h with 1×SSC and 0.1% SDS at 55° C., preferably at 62° C. and more preferably at 68° C., particularly after washing for 1 h with 0.2×SSC and 0.1% SDS at 55° C., preferably at 62° C. and more preferably at 68° C., a hybridization signal is detected.

The degree of identity of the nucleic acid is at least 70%, preferably at least 80%, more preferably at least 90%, and most preferably at least 95% to a reference sequence, e.g. SEQ ID NO: 1, 3 or 5.

Further, the nucleic acid encoding a cytotoxic polypeptide can comprise a partial sequence of the nucleotide sequence as disclosed in SEQ ID NO: 1, 3, or 5. Preferably, the partial sequence is selected from nucleotide No. 115 to 231 in SEQ ID NO: 1, or nucleotide No. 112 to 228 in SEQ ID NO: 3, or nucleic acid residue No. 61 to 177 in SEQ ID NO: 5, or the partial sequence codes for at least one of the eleven fragments of APIT obtained by peptide mass fingerprinting, ESI/MS, and Edman degradation. Further, the partial sequence can comprise a stretch of additional nucleotides selected from the sequences adjacent to the sequence selected from SEQ ID NO: 1, 3, or 5. Preferably, 1-60 additional nucleotides can be present at the 5′ and/or the 3′-terminus. More preferably, 1-30 additional nucleotides can be present at the 5′ and/or the 3′-terminus. Most preferably, 1-10 additional nucleotides can be present at the 5′ and/or the 3′-terminus.

Furthermore, the nucleic acid may encode a fusion polypeptide as described above.

In a preferred embodiment of the invention the nucleic acid is operatively linked to an expression control sequence, e.g. a sequence which is capable of directing expression in a suitable host cell, e.g. a prokaryotic or eukaryotic host cell. The expression control sequence usually comprises a promoter and optionally operator or enhancer sequences which enable a transcription of the nucleic acid operatively linked thereto. Furthermore, the expression control sequence may contain a translation signal, e.g. a ribosome binding sequence.

The nucleic acid of the present invention may be a recombinant vector which contains in addition usual vector sequences such as an origin of replication, a selection marker gene and/or a cloning site. Examples of suitable vectors such as plasmids, phages or viral vectors are known to the skilled person and are described e.g. in Sambrook et al., Molecular Cloning, A Laboratory Manual (2nd ed. 1998), Cold Spring Harbor, Laboratory Press.

A further aspect of the present invention is a recombinant cell transformed or transfected with a nucleic acid as described above. The recombinant cell may be a prokaryotic cell, e.g. a gram-negative prokaryotic cell such as E. coli or an eukaryotic cell, e.g. an insect cell or a vertebrate cell such as a mammalian cell. Techniques for transforming or transfecting host cells with nucleic acids are known to the skilled person and e.g. described in Sambrook et al., supra.

Still a further subject matter of the present invention is an antibody directed against the polypeptide as described above. The antibody may inhibit the cytotoxic activity of the polypeptide. The antibody may be a polyclonal or monoclonal antibody or a recombinant antibody, e.g. a chimeric antibody, a humanized antibody or a single chain antibody. Furthermore, the antibody may be an antibody fragment containing the antigen-binding site of the antibody, e.g. a Fab fragment. The antibody may be obtained by immunizing suitable experimental animals with an Aplysia polypeptide as described above or a partial fragment thereof or a peptide antigen optionally coupled to a suitable macromolecular carrier according to known protocols, e.g. by techniques which are described in Borrebaeck, Carl A. K. (Ed.), Antibody engineering (1992), or Clark, M. (Ed.), Protein engineering of antibody molecules for prophylactic and therapeutic applications in man (1993). By techniques for producing hybridoma cell lines according to Köhler and Milstein monoclonal antibodies may be obtained.

Methods for introducing a prosthetic group into a polypeptide are known in the art. Preferably, the FAD is introduced by a method comprising surface display of the polypeptide on a prokaryotic host, comprising the steps:

-   -   (a) providing a prokaryotic host cell transformed with a nucleic         acid fusion operatively linked with an expression control         sequence, said nucleic acid fusion comprising sequences         necessary for displaying the protein on the outer membrane, and     -   (b) culturing the host cell under condition wherein the nucleic         acid fusion is expressed and the expression product comprising         the recombinant polypeptide is displayed on the surface of the         host cell, and     -   (c) contacting the recombinant polypeptide with FAD under         conditions wherein FAD combines with the recombinant polypeptide         and a functional recombinant polypeptide containing the         prosthetic group is formed.

The nucleic acid fusion may be formed using a nucleic acid sequence as described above and further sequences necessary for surface display. Details describing the prokaryotic host cells, the sequences necessary for surface display of the polypeptide, culture conditions, and the conditions under which the recombinant polypeptide is contacted with FAD are described in WO 02/070645, which is included by reference herein.

A further aspect of the present invention relates to diagnostic or therapeutic applications in humans or animals. The polypeptide, and/or a nucleic acid, and/or a recombinant cell, and/or an effector, e.g. an inhibitor or activator of the polypeptide as described above can be used in such applications. The polypeptide as described above is able to selectively kill tumor cells. For example, T and B leukemia cell lines, a chronic myeloid leukemia cell line (K562), cells from an orphan and aggressive osteosarcoma (Ewings tumor: RDES, A673), a small cell lung cancer cell line (GLC4, GLC4/ADR), cervix cancer (Chang), and acute monocytic leukemia (THP-1) show an IC₅₀≦10 ng/ml APIT.

Healthy human cells are resistant against APIT-induced cell death. At a concentration of 40 ng/ml, APIT induces a cell death below 10% in normal HUVEC cells (Example 13). This indicates that the APIT IC₅₀ values of healthy cells are at least one order of magnitude higher than the IC₅₀ of tumor cells.

Resistance to apoptosis as well as multi drug resistance (MDR) represent severe problems in cancer therapy. It is therefore of particular interest that the polypeptide of the present invention kills apoptosis resistant cell lines as well as MDR cancer cell lines to the same extent as their non resistant counter parts. Over-expression of apoptosis inhibitors of the Bcl-2 family in cancer cell lines does not protect from APIT mediated cell death, confirming that APIT induces cell death in an apoptosis independent way. The MDR cell line GLC4/ADR possess almost the same sensitivity to APIT IC₅₀ 10 ng/ml) as the parental cancer line GLC4 does IC₅₀ 9 ng/ml).

Thus, the diagnostic or therapeutic application preferably relates to a method for diagnosis or treatment of hyperproliferative diseases, e.g. cancer. More preferably, the method is a method for diagnosis or treatment of lung cancer, breast cancer, prostate cancer, colon cancer, cervix cancer, uterus cancer, larynx cancer, stomach cancer, liver cancer, Ewings sarkoma, acute lymphoid leukemia, acute and chronic myeloid leukemia, apoptosis resistant leukemia, and/or MDR lung cancer. Moreover other tumor types can also be treated with the polypeptide, like pancreas cancer, gastric cancer, kidney cancer, gliomas, melanomas, chronic lymphoid leukemia, and/or lymphoma. Since all cancer cell lines tested (in total 24) were effectively killed by APIT, the polypeptide can be used for the treatment of solid tumors and leukemias in general including apoptosis resistant and multi drug resistant cancer forms.

A further aspect of the present invention is a pharmaceutical composition comprising the polypeptide of the present invention as described above, in a pharmaceutically effective amount and optionally together with suitable diluents and carriers or kit containing the composition together with other active ingredients, e.g. modulators of the polypeptide or other cytostatic or cytotoxic agents. The composition can be administered locally or systemically by any suitable means, e.g. orally, nasally or by injection (i.v., i.p., s.c., or i.m.) to a subject in need thereof. The components of a kit, which consists of at least two different compositions may be administered together or separately, e.g. at different times and/or by different routes.

In another embodiment, the pharmaceutical composition or the kit comprises a nucleic acid encoding for the polypeptide of the present invention as described above. Further, the pharmaceutical composition or kit may comprise both the polypeptide and the nucleic acid of the present invention. From many studies it is known that tumor cells have an increased rate of metabolism compared to normal cells. A result of this high metabolic rate is a high concentration of reactive oxygen species (ROS, comprising H₂O₂) which originate from oxidative phosphorylation reactions by the electron transport chain of the mitochondria. As a consequence ROS detoxification reactions are increased in tumor cells, and interference with detoxification has a selective toxic effect on the tumor cells but not on normal cells. Likewise, increasing the concentration of H₂O₂ by administering the polypeptide of the invention in a predetermined amount may overcome the detoxification reactions and kill the tumor cells. The level of extra H₂O₂ produced by exogenous APIT does not affect normal cells because of their higher tolerance for additional H₂O₂. An administration of the polypeptide in a varying amount, e.g. a gradually changing, e.g. increasing amount leads to the production of a defined amount of H₂O₂ could thus be used for a selective killing of cancer cells.

The pharmaceutical composition or kit as described above can comprise a further component which is a substance capable of modulating the cytotoxic activity of the polypeptide, in a pharmaceutically effective amount and optionally together with suitable diluents, and carriers. In FCS (100%) at 37° C. and 5% CO₂ which reflect in vivo conditions, or in a medium containing 10% FCS (typical in vitro conditions) devoid of L-lysine and L-arginine, the activity of APIT (20 ng/ml) can be dose-dependently increased by the addition of L-lysine in a final concentration of 2-50 μg/ml. Thus, the high specifity of APIT for L-lysine (and L-arginine) allows for modulating the enzymatic activity of APIT and thus its cytotoxic activity by providing an additional substrate in vivo or in vitro. The substance capable of modulating the cytotoxic activity of the polypeptide can be L-lysine, L-arginine, a derivative or metabolic precursor of L-lysine, or L-arginine, or a mixture thereof. A derivative is a compound which is an APIT substrate. A metabolic precursor is a compound, which can be metabolized to a compound, which is an APIT substrate. Further, the modulator may be selected from flavine nucleosides, particularly FAD, since the presence of a flavine nucleoside prosthetic group leads to a great increase in APIT activity.

The pharmaceutical composition may comprise the polypeptide and at least one modulating substance as a mixture. Preferably, the modulating substances are provided in a kit consisting of separate preparations. More preferable, the polypeptide is provided for administration before the modulating substances.

During the passage through body fluids before reaching the tumor tissue, the cytotoxic activity of the polypeptide would be undesired, due to the toxic properties of H₂O₂. Thus, the composition may further comprise an inhibitor of the polypeptide. The inhibitor could have a short half-life time in the body fluid. A preferred inhibitor of the polypeptide is an antibody against the polypeptide (see above).

Modulating the activity of the polypeptide of the present invention can also be accomplished by modulating the product level, i.e. the H₂O₂ level. The degradation of at least one of the products, namely H₂O₂, results further on in consumption of the substrates L-lysine and L-arginine by the polypeptide of the present invention. Thus, these amino acids may be deprived. Since L-lysine and L-arginine are essential for living and growing of tumor cells, deprivation of these amino acids by a combination of the polypeptide of the present invention and an H₂O₂ scavenger may lead to the death of tumor cells. Thus, in another embodiment, the pharmaceutical composition may comprise the polypeptide of the present invention and an H₂O₂ scavenger. A preferred H₂O₂ scavenger is catalase. Preferably, a kit is provided consisting of separate preparations of the polypeptide of the present invention and catalase.

Further the polypeptide can be coupled with a substance and/or a particle which targets the polypeptide to the tumor tissue.

Further components of the pharmaceutical composition can be a nucleic acid coding for the polypeptide as described above, and/or a recombinant vector or cell containing the nucleic acid.

A further aspect of the present invention is a substance modified by interaction with APIT (termed target substance of APIT). A direct interaction is a contact of APIT with this substance. In an indirect interaction, the effect upon the substance includes at least one mediator substance, e.g a substance formed by APIT, or a receptor interacting with APIT and the components of the related transduction cascade.

As described above, a mediator of APIT acting on cellular polypeptides is H₂O₂. Thus, preferred target substances of APIT comprise cellular polypeptides, which can be modified by H₂O₂. A major modification identified in 2-DE SDS gel patterns of cells treated with APIT was a shift of peroxiredoxin I (Prx I, Swiss-Prot No. Q06830, Genbank identifier No. 548453, SEQ ID NO: 8), which was also detected in cells treated with H₂O₂. Prx I belongs to a class of peroxidases which are involved in the detoxification of ROS. Although the nature of the modification of Prx is not known, Prx I can be used as a marker for APIT anti-tumor activity.

Thus, particularly preferred substances which can be used as target substances of the polypeptide as described above are peroxidases, especially preferably peroxiredoxin I or a polypeptide having substantially the same biological activity as peroxiredoxin I. Peroxiredoxin I may comprise

-   -   (a) the amino acid sequence shown in SEQ ID NO: 8, or/and     -   (b) an amino acid sequence which is homologous to the sequence         of (a) with at least 70%, preferably 80%, particularly         preferably 90%, especially preferably 95%, or/and     -   (c) a fragment of the amino acid sequence of (a) or (b).

Further, peroxiredoxin I may comprise an amino acid sequence or a fragment thereof as disclosed in at least one of the Genbank entries selected from gi:4505591 (NP_(—)002565.1), gi:13626803 (XP_(—)001393.2), gi:32455264 (NP_(—)859047.1), gi: 32455266 (NP 859048.1), gi: 423025 (A46711), gi: 287641 (CAA48137.1), gi: 13937907 (AAH07063.1), gi: 18204954 (AHH21683.1) or gi:440306 (AAA50464.1).

WO 02/31144 discloses proteins modified by H₂O₂ which are targets of APIT: thioredoxin peroxidase 2 (Swiss Prot No. Q06830, Genbank identifier 548453), 60S ribosomal protein P0 (12654583), Hsp-60 (N-term) (14603309), stathmin (5031851), Rho GDI 2 (P52566, 1707893), 60S ribosomal protein P0(4506667), RNA binding regulatory subunit (O14805,12720028), hnRNP C1/C2 (4758544), hnRNP C1/C2 (4758544), proteasome subunit beta type 1 (P20618, 130853), pre-mRNA cleavage factor Im (5901926), proteasome subunit alpha type 7 (O14818,12643540), U2 small nuclear ribonucleo-protein A′ (P09661,134094), GAP SH3 binding protein (5031703), DNA replication licensing factor MCM4 (P33991, 1705520), thioredoxin peroxidase 1 (P32119,2507169), 40S ribosomal protein S21 (P35265, 464710),40S ribosomal protein S12 (P25398,133742), phosphoglycerate mutase 1 (P18669,130348), HCC-1 protein (13940310), HnRNP A2/B1 (4504447/14043072), IMP dehydro-genase 2 (P12268, 124419), hnRNP A/B (14724990).

Further targets of APIT identified by 2 DE gel electrophoresis, in-gel tryptic digestion, peptide mass fingerprinting by MALDI-MS, and identification of the proteins are summarized in Table 3.

Still a further target of APIT is a nucleic acid. The target nucleic acid can be a DNA or an RNA, which is a mRNA. The transcription of the mRNA is up- or downregulated in the presence of APIT and/or H₂O₂. Preferably, the transcription is changed by a factor of at least 2, and more preferably, by a factor of at least 4.

By a microarray of specific 60 mer oligonucleotides representing about 8500 human genes, about 70 mRNAs were identified which are targets of APIT. The information about the mRNAs are summarized in Table 4. Each mRNA is referenced by a “unigene cluster” which represents a number of nucleotide sequences belonging to the same gene or to closely related genes. Details of the nomenclature and the nucleotide sequences of the unigene clusters are public available under available via the world wide web at ncbi.nlm.nih.gov/ (Homepage of the National Center for Biotechnology Information).

For most of the unigene clusters of Table 4, the gene and/or the protein is known. It is a general principle that modulation of the transcription of a messenger RNA influences the amount of protein expressed. Thus, the proteins coded by the sequences of the unigene clusters of Table 4 are also targets of APIT, because APIT may influence their expression. The sequences of the proteins and of the nucleic acids coding for these proteins are referenced by the genbank identifier, accession number and/or version number (see Table 4). The sequences are public available under available via the world wide web at ncbi.nlm.nih.gov/.

Additional targets of APIT (nucleic acids, proteins) obtained by microarray analysis as described above are summarized in Table 5.

A preferred substance which can be used as a target substance for the polypeptide as described above is a nucleic acid coding for a peroxidase, particularly preferably peroxiredoxin I or a polypeptide having substantially the same biological activity as peroxredoxin I. The nucleic acid coding for peroxiredoxin I may comprise

-   -   (a) the nucleotide sequence shown in SEQ ID NO: 7, or/and     -   (b) a nucleotide sequence which corresponds to the sequence         of (a) within the scope of the degeneracy of the genetic code,         or/and     -   (c) a nucleotide sequence hybridizing to the sequence of (a)         or/and (b) under stringent conditions, or/and     -   (d) a fragment of the nucleotide sequence of (a), (b) or (c).         SEQ ID NO: 7 is disclosed in Genbank entry gi:14721336         (XM001393).

Preferably, the nucleic acid encoding peroxiredoxin I may comprise a nucleotide sequence which is homologous to SEQ ID NO: 7 with at least 70%, particularly preferably at least 80%, especially preferably at least 90%.

In further preferred embodiments, the nucleic acid encoding peroxiredoxin I may comprise a nucleotide sequence or a fragment thereof as disclosed in at least one of the Genbank entries selected from gi: 13937906 (BC007063.1, PRDX1 transcript 3), gi: 18204953 (BC021683.1, PRDX1 transcript variant 3), gi: 32455265 (NM_(—)181697.1, PRDX1 transcript variant 3), gi: 34528302 (AK131049.1, clone highly similar to PRDX1), gi: 287640 (X679851.1, PAG), gi: 32455263 (NM_(—)181696.1, PRDX1 transcript variant 2), gi: 32455267 (NM_(—)002574.2, PRDX1 transcript variant 2) or gi:440305, (L19184, NKEF A).

The target substance of the present invention (see Table 3, 4 and 5), which is identified by one of the methods as described above, may be used for the development of new pharmaceutical agents, e.g. by known high-throughput screening procedures which may be cellular screening procedures or molecular based screening procedures. These pharmaceutical agents may act upon cellular receptors and/or components of the signal transduction pathways activated or inhibited by APIT.

Degenerative diseases like Alzheimer's and Parkinson's disease are characterised by excessive ROS production of the affected tissue. Drugs which either activate H₂O₂ detoxification or inhibit H₂O₂ production may be used for therapy of degenerative diseases like Alzheimer's or Parkinson's disease. Fast growing tumor cells produce more ROS and thus require an efficient H₂O₂ detoxification system. Drugs which either activate H₂O₂ production or which interfere with H₂O₂ detoxification may be used for therapy of proliferative diseases like tumors. Since e.g. thioredoxin peroxidases 1 and 2 have been shown to be overexpressed in cells at risk for diseases related to ROS toxicity including degenerative diseases like Alzheimer's and Parkinson's disease, and have been shown to be overexpressed in tumor cells (Butterfield et al., 1999, Antioxidants & Redox Signalling, 1, 385-402), the targets of Table 3 and 4 might be important targets for the development of drugs for treatment of degenerative diseases like Alzheimer's and Parkinson's disease and of proliferative diseases like tumors.

NK-cells have been shown to protect against malignant cells in chronic myelogenous leukemia (CML), but their number and inducibility is reduced during the progression of the disease. This reduction and dysfunction is due to the production of H₂O₂ by CML-cells (Mellqvist, Blood 2000, 96, 1961-1968). NK-cells encountering H₂O₂ are inhibited in their Iytic activity, are made resistant to IL-2 activation and undergo apoptosis/necrosis. Any therapy providing CML-patients with ROS-hyposensitive NK-cells therefore would be of great benefit. The targets described above could be used to modulate the H₂O₂ sensitivity of NK-cells or to inhibit the H₂O₂ production of malignant cells, e.g. CML-cells.

Arteriosclerosis with its progression to heart disease, stroke and peripheral vascular disease continues to be the leading cause of death in all western civilisations. Enhanced ROS-production (via endothelial NADPH-oxidase) is required and sufficient to generate the pathologic phenotype (Meyer, FEBS Letters 2000, 472, 1-4). Therefore, targets mediating the effect of H₂O₂ are useful to develop new drugs for treatment of arteriosclerosis and the associated diseases like heart disease, stroke and other vascular diseases. These targets are suitable to detoxify H₂O₂ and/or to block the H₂O₂ induced signalling pathways.

Target compounds, e.g. peptides, polypeptides or low-molecular weight organic compounds, which are capable of modulating the effect of H₂O₂ may be identified in a screening system comprising the use of the APIT polypeptide as described above. Particularly, a modulation of the APIT activity, i.e. L-amino oxidase activity, may be determined.

Thus the present invention further relates to a pharmaceutical composition comprising as an active agent at least one of the target substances as described above.

Still a further aspect of the present invention is an inhibitor of a target as described above, in particular an inhibitor of the detoxification system of the cell which eliminates reactive oxygen species, e.g. H₂O₂. Surprisingly, it was found that the inhibition of detoxifying enzymes sensitized tumor cells to the cytotoxic activity of the polypeptide of the present invention as described above. Example 11 demonstrates that knock-down of peroxredoxin I sensitized tumor cells for APIT-induced cell death.

Preferably, the inhibitor is an inhibitor of peroxidase, particularly of peroxiredoxin I. The inhibitor may be an antibody or a nucleic acid molecule, i.e. useful for antisense inhibition or as an siRNA molecule. It is particularly preferred that the inhibitor is an inhibitor of peroxiredoxin I activity which is an RNA molecule, particularly a double-stranded RNA molecule comprising a nucleic acid sequence of at least 15 nucleotides complementary to a peroxiredoxin I transcript. It is especially preferred that the peroxiredoxin I transcript is derived from SEQ ID NO:7.

The one or two strands of the RNA molecule as described above may, independently, have a length of 19 to 25 nucleotides, preferably 19 to 23 nucleotides. Especially preferred is a length of the one or two strands of 19, 20, 21, 22 or 23 nucleotides. The RNA molecule as described above may comprise at least one modified nucleotide. Preferably, modified nucleotides are selected from the group consisting of oxetane[1-(1′,3′-O-anhydro-β-D-psicofuranosyl)-nucleotides, locked nucleic acid (LNA) nucleotides, hexitol nucleotides, altritol nucleotides, cyclohexane nucleotides, neutral phosphatate analogs.

The double-stranded RNA molecule as described above may have one or two 3′ overhangs with, independently, a length of 1 to 5 nucleotides, preferably 1 to 3 nucleotides, particularly preferably 2 nucleotides. The one or two overhangs may consist of ribonucleotides, deoxyribonucleotides, modified nucleotides as described above or combinations thereof.

The double-stranded RNA molecule as described above may comprise a sequence selected from the group of sequences consisting of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11; SEQ ID NO: 12; SEQ ID NO: 13; SEQ ID NO: 14; SEQ ID NO: 15; SEQ ID NO: 16; SEQ ID NO: 17; SEQ ID NO: 18; SEQ ID NO: 19; SEQ ID NO: 20; SEQ ID NO: 21; SEQ ID NO: 22; SEQ ID NO: 23; SEQ ID NO: 24; SEQ ID NO: 25; SEQ ID NO: 26; SEQ ID NO: 27; SEQ ID NO: 28; SEQ ID NO: 29.

Yet another aspect of the present invention is a pharmaceutical composition or kit comprising an inhibitor as described above, preferably an RNA molecule, particularly preferred a double-stranded RNA molecule, or a nucleic acid encoding such an RNA molecule. The pharmaceutical composition or kit may comprise the inhibitor as sole active agent in order to increase the amount of reactive oxygen species present in the cell due to endogenous production. More importantly, the pharmaceutical composition or kit may comprise the inhibitor and a substance capable of producing reactive oxygen species. In a preferred embodiment, the pharmaceutical composition or kit comprises as an active agent a combination of APIT and at least one inhibitor of a target substance as described in Table 3 or/and Table 4 or/and Table 5, more preferably at least one inhibitor of peroxiredoxin I. In another preferred embodiment, the pharmaceutical composition or kit comprises at least one inhibitor of a target substance as described in Table 3 or/and Table 4 or/and Table 5, more preferably at least one inhibitor of peroxiredoxin I, and the polypeptide of the present invention having cytotoxic activity as described above. In yet another preferred embodiment, the pharmaceutical composition or kit comprises at least one inhibitor of a target substance as described in Table 3 or/and Table 4 or/and Table 5, more preferably at least one inhibitor of peroxredoxin I, and a cytotoxic polypeptide producing reactive oxygen species or/and a nucleic acid encoding such a cytotoxic polypeptide, wherein the cytotoxic polypeptide is selected from cytotoxic polypeptides obtainable from sea hares, e.g. Cyplasin C, Cyplasin L, Aplysianin A, Aplysianin P, Aplysianin E, Dolabellin A, Dolabellin C, Dolabellin P, Julianin G, Julianin S, or is selected from L-Lysine oxidases like EC 1.4.3.14 from Trichoderma, AIP from Chub mackerel (AJ400871), Apoxin from Crotalus (AAD45200.1), or from other L-amino acid oxidases like EC 1.4.3.2 or from other enzymes which produce H₂O₂. More preferably, the pharmaceutical composition or kit comprises

(I) a polypeptide obtainable from Aplysia comprising an amino acid sequence selected from:

(a) D-G-E-D-A-A-V (SEQ ID NO:32) and/or (b) (D/Q)-G-(I/V)-C-R-N-(Q/R)-R-(Q/P), (SEQ ID NO:33) (c) F-A-D-S, (SEQ ID NO:34) (d) G-P-D-G-(I/L)-V-A-D, (SEQ ID NO:35) (e) P-G-E-V-S-(K/Q)-(I/L), (SEQ ID NO:36) (f) A-T-Q-A-Y-A-A-V-R-P-I-P-A-S-K, (SEQ ID NO:37) (g) D-S-G-L-D-I-A-V-E-Y-S-D-R, (SEQ ID NO:38) (h) G-D-V-P-Y-D-L-S-P-E-E-K (SEQ ID NO:39) or/and (i) SEQ ID NO:41, 43, 44, 45. or a fragment thereof wherein the polypeptide or the fragment has cytotoxic activity, or/and a nucleic acid encoding the cytotoxic polypeptide obtainable from Aplysia comprising

-   (i) a nucleotide sequence as shown in SEQ ID NO:40 or 42 or at least     the polypeptide coding portion thereof or the complement thereof, -   (ii) a nucleotide sequence corresponding to the sequence of (a)     within the scope of degeneracy of the genetic code, or the     complement thereof, or/and -   (iii) a nucleotide sequence hybridizing under stringent conditions     with the sequence of (a) or/and (b), and     (II) an inhibitor of a target substance as described in Table 3     or/and Table 4 or/and Table 5.

The inhibitor of the present invention may be coupled to carriers, (e.g. lipids, peptides, biodegradable polymers, dendrimers, vitamins, carbohydrate receptors) for in vivo targeting to predetermined tissues or/and cell types.

Delivery of the inhibitors of the present invention may be improved by linking the inhibitors with lipids, liposomes, PEG, nanoparticles or/and polymers, for example.

Yet another aspect of the present invention is a gene therapy delivery system suitable for delivery of a nucleic acid encoding an inhibitor which is an RNA molecule, preferably a double-stranded RNA molecule as described above, capable of inhibiting peroxidase, particularly peroxiredoxin I activity. Suitable delivery systems for gene therapy are commonly known in the art, for instance a recombinant adenoviral delivery system, a recombinant adenoviral-derived system or a recombinant lentiviral system. Further, the nucleic acid may be delivered by virus-like particles from Papillomaviridae and Polyomaviridae. Further, bacteria may be used as a delivery system, e.g. attenuated gram negative bacteria, particularly attenuated salmonella strains. The nucleic acid encoding the inhibitor is operatively linked with expression control sequences which are adapted to the host and to the delivery system. Such expression control sequences are known to a person skilled in the art. Expression of the two strands of the RNA molecule may be performed together in a self-complementary configuration which allows formation of a small hairpin RNA (shRNA) in which the two strands of the double-stranded molecule are interconnected by an additional loop, or may be performed as two separate strands which hybridize later on in the host.

Yet another aspect is a pharmaceutical composition or kit comprising a delivery system suitable for delivery of a nucleic acid encoding an inhibitor which is an RNA molecule, particularly a double-stranded RNA molecule preferably comprising a nucleic acid of at least 15 nucleotides complementary to a peroxiredoxin I transcript as described above, to predetermined tissues or/and cell types.

In yet another embodiment, the invention concerns a method for diagnosis or treatment of cancer, wherein a pharmaceutical composition as described above is administered to a subject in need thereof.

SEQ ID NO: 1, 3 and 5 show the APIT nucleotide sequences as shown in FIG. 4 c. SEQ ID NO: 2, 4 and 6 show the amino acid sequences derived from SEQ ID NO: 1, 3 and 5, respectively. SEQ ID NO: 7 and 8 show the nucleotide sequence and the amino acid sequence of Prx I. SEQ ID NOs: 9 to 29 show the nucleotide sequences of double-stranded siRNA molecules capable of inhibiting Prx I activity. SEQ ID NOs: 30 and 31 show sequences of double stranded siRNA molecules obtained from the Lamin AC and the luciferase sequence, respectively. SEQ ID NOs: 32 to 39 show the amino acid sequences of fragments of cytotoxic Aplysia polypeptides. SEQ ID NO: 40 and 42 show partial sequences of nucleic acids encoding cytotoxic polypeptides of Aplysia punctata. SEQ ID NOs: 41,43,44 and 45 show the derived amino acid sequences of SEQ ID NOs: 40 and 42. SEQ ID NOs: 46 to 55 show the amino acid sequences of fragments of cytotoxic Aplysia polypeptides.

The invention is explained in more detail by the following figures, tables and examples.

FIG. 1

A, Anion exchange chromatography. Filtrated and concentrated ink was loaded onto a Source Q15 column. Proteins were eluted by a linear gradient from 0 to 800 mM NaCl, fractions were collected every minute (2 ml/min). Absorption was measured at 280 nm. Horizontal bar indicates active fractions.

B, Gelfiltration. Active fractions from the Source Q15 were pooled and concentrated and applied to a Superose 12 HR 10/30 column. Proteins were eluted with 100 mM potassium phosphate buffer (pH 7.2). Fractions were collected every minute (0.5 ml/min). Horizontal bar indicates active fractions.

FIG. 2

A, Phenotype of APIT-induced cell death. Jurkat cells were cultured for 7 hours in the presence (APIT) or absence (medium) of APIT (30 ng/ml) and phase contrast images were recorded.

B, Lack of apoptotic DNA fragmentation in ink-treated cells. Jurkat cells were incubated in medium (control) or treated with cycloheximide (chx; 10 μg/ml) or ink (ink, 1/500 diluted) for 2, 4 and 6 h. Isolated DNA was visualized on a 1,6% agarose gel by ethidium bromide staining.

C, APIT mediated loss of metabolic activity. APIT (10 ng/ml) and the tetrazolium-salt WST-1 were added simultaneously to Jurkat cells and turnover of WST-1 was measured photometrically. White circles: medium control; black circles: APIT-treated samples; mean absorbance of 8 replicates ″SD.

D, Cell death induced by ink. Jurkat cells were treated with ink (1/500 diluted) and propidium iodide (PI) uptake was measured as indicator for dead cells.

FIG. 3

A, Heat sensitivity of ink. Dialysed ink was incubated for 10 min at the indicated temperatures and enzymatic activity was measured as H₂O₂-production (mean of triplicates ″SD). Blank: medium control.

B, pH-sensitivity of APIT. APIT (60 ng) was incubated for 10 min at 25° C. in 0.1 M potassium phosphate at indicated pH values. Enzymatic activity was measured as H₂O₂-production (mean of triplicates ″SD).

C, Sensitivity to increasing amounts of urea: Dialyzed ink (black bars, 1/500 diluted) and as positive control 0.625 mM á-keto isocaproic acid (open bars) were treated with indicated concentrations of urea for 30 min at 25° C. Enzymatic activity (15 min. 25° C.) was measured as á-keto acid formation via MBTH.

FIG. 4

A, N-terminal (SEQ ID NO: 33) and internal peptide sequences (SEQ ID NOS 47,49-50, 77, 51, 37, and 52-55 disclosed respectively in order of appearance) of the APIT protein.

B, List of oligonucleotides used for cloning of the APIT gene (SEQ ID NOS 58-61, 63, 62, and 64-66 disclosed respectively in order of appearance).

C, Nucleotide sequence of the APIT CDNA (SEQ ID NO: 1) and the derived amino acid sequence (SEQ ID NO: 2). The dinucleotide binding fold (VAVVGAGPGGANSAYMLRDSG-LDIAVFE) (SEQ ID NO: 56) and the GG-motif (RVGGRLFT) (SEQ ID NO: 57) are indicated by boxes. Consensus amino acid residues are indicated by bold letters. The N-terminal sequence of mature APIT (dashed line) and of internal peptides (solid line) derived by Edman degradation and mass finger prints are indicated. Sequence variations of the three clones are indicated by small boxes (SEQ ID NOS 3 and 5 are also disclosed respectively in order of appearance).

D, Variation of the N-terminus of APIT in 11 further clones (SEQ ID NO: 78).

FIG. 5

A, Anion exchange chromatography of purified APIT. Proteins were eluted by a linear gradient from 0 to 800 mM NaCl and fractions were collected every minute. Absorption was measured at 280 nm (AU: Absorption unit).

B, Fractions 24,27 and 29 were separated by SDS-PAGE and tested for metabolic activity by WST-1 assay. High activity (+;++) correlated with the presence of a prominent 60 kDa band (fractions 24 and 29). Activity is given as the dilution leading to >85% reduction of the metabolic activity of Jurkat cells (+/−=1:900; +=1:2700; ++=1:8100).

C, Absorption spectra of fractions 24 (black line), 27 (dashed line) and 29 (dotted line).

FIG. 6

A, APIT induced H₂O₂ production in medium in the absence of cells. APIT (260 ng/ml) was incubated in medium in the presence (open bar) or absence (black bar) of Jurkat cells (5×10⁵/ml). After 1 h of incubation at 37° C. supernatants were alkylated with N-ethylmaleimide and H₂O₂ was measured (mean values of 3 independent experiments +/−SD).

B, Catalase inhibits ink induced cell death. Jurkat T-cells were incubated for 8 h with ink in the presence (black bars) or absence (white bars) of catalase. Cytotoxicity was measured as PI uptake (mean of triplicates ″SD).

C, Catalase protects from APIT induced loss of metabolic activity. Metabolic activity of Jurkat cells was measured after incubation with APIT (20 ng/ml) or anti-CD95 for 3 h in the presence (black bars) or absence (white bars) of catalase. (mean of 5 replicates ″SD).

D, Phenotype of APIT induced cell death is mediated by hydrogen peroxide. Jurkat cells were cultured for 7 hours in the presence (APIT) or absence (medium) of APIT (60 ng/ml) or H₂O₂ (500 μM) and were analyzed by phase contrast microscopy. Catalase was added in combination with APIT to neutralize H₂O₂ (APIT+CAT).

E, Long-term exposure with ink from Aplysia punctata and catalase resulted in tumor cell death by amino acid deprivation. Metabolic activity of Jurkat T-cells was measured after overnight incubation (>18 h) with ink (white bars) or H₂O₂ (250 iM, black bars) in the presence (+) or absence (−) of catalase (2000 U/ml) (mean of triplicates ″SD).

FIG. 7

A, Enzymatic activity of APIT in the presence of different medium supplements. APIT (200 ng/ml) was incubated for 60 min at RT with RPMI +/−10% FCS or KRG supplemented with different medium ingredients and H₂O₂ production was measured. (EAA=essential amino acids, NEAA=non essential amino acids, concentrations see Table 1).

B, Substrate specificity of APIT and ink. The enzymatic reaction of dialysed ink (open bars) with different L-amino acids in potassium phosphate buffer was measured as H₂O₂-production. 50 μM H₂O₂ and amino acid free medium (control) were used as control. Aliquots of dialyzed ink were digested with trypsin (hatched bars) or proteinase K (black bars) at 37° C. for 2 h prior to testing the substrate specificity. Arg=L-arginine, 1 mM; Lys=L-lysine, 1 mM; EAA essential amino acids, 1 mM; NEAA=non essential amino acids, 1 mM.

C, APIT induced cell death depends on the presence of L-lysine or L-arginine. Jurkat cells were incubated with APIT (20 ng/ml) for 6 h in the presence (white bars) or absence of L-lysine and L-arginine (black bars). Cytotoxicity was measured as PI uptake (mean of triplicates ″SD).

D, APIT induced loss of metabolic activity depends on the presence of L-lysine or L-arginine. Jurkat cells were incubated with APIT (20 ng/ml) or anti-CD95 (150 ng/ml) in the presence (open bars) or absence (black bars) of L-lysine or L-arginine and metabolic activity was measured (mean of 5 replicates ″SD).

E, APIT transforms L-lysine into an á-keto acid. APIT was incubated with L-lysine and the formation of á-keto acid was measured photometrically by its reaction with MBTH.

F, Michaelis-Menten kinetic of APIT activity with L-lysine. K_(m) value for L-lysine was determined as H₂O₂ production.

G, Proposed reaction mechanism of L-amino acid oxidases according to Macheroux et al. (2001 Eur. J. Biochem. 268:1679-1686). Encircled are compounds which we demonstrated to participate in the reaction catalyzed by APIT.

FIG. 8

A, Quantification of the mRNAs of Lamin A/C and Prx I after transfection of specific siRNA (open bars) and control Luciferase siRNA (black bars) with quantitative realtime PCR. Shown are the relative mRNAs levels compared to the mRNA of GAPDH measured in the same RNA preparation.

B, Sensitization of HeLa cells by knock down of Prx I. Specific siRNAs directed against the mRNA of Luciferase (Luc, transfection control), Lamin A/C (control knock down) and Prx I were transfected in HeLa cells and the metabolic activity of transfectants treated in the presence (black bars) or absence of APIT (open bars) was measured. Note that the knock down of Prx I but not of the other genes sensitized cells for the cytotoxic activity of APIT.

FIG. 9

APIT did not induce actin depolymerisation in HeLa cells. Untreated HeLa cells (A) and HeLa cells treated with Cytochalasin (B) or APIT (C) were stained with Phalloidin-TRITC for actin and Hoechst 33258 for nuclei staining. Subsequently, fluorescence microscopy was performed. Actin staining is shown in bright white, nuclei are displayed in transient grey.

FIG. 10

HUVEC cells are resistant to the APIT induced cells death. HUVEC and Jurkat cells were incubated with APIT over night and subsequently LDH release in the culture supernatant was measure photometrically. Shown are the results of two independent experiments+/−standard deviation.

TABLE 1

Composition and concentrations of mixtures of essential and non-essential amino acids as well as single amino acids used in FIG. 7A.

TABLE 2

APIT kills different kinds of tumor cells. Different tumor cell lines (50,000 cells in 100 Fl) were incubated for 14 h in the presence of increasing amounts of APIT. Metabolic activity of the cells was measured via turnover of WST. The IC₅₀ values reflect the APIT concentration at which the metabolic activity is decreased to 50%. (* stands for IC₅₀≧20 ng/ml at the given cell concentration of 50,000/100 Fl.)

TABLE 3

List of proteins which were changed in their expression or modified after treatment with APIT (upregulation (+), downregulation (−), or modification (m) in column “effect”). The proteins are referenced by the genbank identifier and/or accession number and/or version number.

TABLE 4 AND TABLE 5

List of genes (referenced by unigene cluster number) and gene products (proteins) which were modulated in their expression more than 2 fold after incubation with APIT for two hours. The proteins are referenced by the genbank identifier and/or accession number. Transcription rates are indicated as increase (+, 2 to ≦4 times; ++, 4 to 6 times in Table 4 or 4 to 25 times in Table 5) or decrease (−, 2 to ≦4 times; −−, 4 to 6 times).

EXAMPLE 1 Purification of APIT

Aplysia punctata were gained from the Station Biologique Roscoff, Bretagne, France. Crude ink was prepared by gentle squeezing the sea hares in sterile seawater. Insoluble particles were removed by ultracentrifugation (82,000 g, 30 min, 4° C.) and supernatants were stored at −70° C.

APIT was purified from crude ink via anion exchange chromatography and gelfiltration. The thawed ink was filtered through Whatman filter No. 4 under slight vacuum and subsequently through a 5 Fm and 0.45 Fm syringe filter. The filtrate was concentrated by using Ultrafree-15 Units (Millipore, exclusion weight 30 kDa) followed by three washing steps with 20 mM Tris HCl (pH 8.2). After centrifugation at 10.000 g for 5 min the supernatant of the concentrate (20-60 fold) was applied to a Source Q15 column ((10 mm, length 40 mm) equilibrated with 20 mM Tris HCl, pH 8.2. Proteins were eluted by a linear gradient from 0 to 800 mM NaCl over 50 ml at a flow rate of 2 ml/min (FIG. 1A). The purity of the fractions was determined by SDS-PAGE and subsequent rapid silver staining. APIT appears as a band at 60 kDa. Cytolytic activity was measured as APIT-induced reduction of the metabolic activity of Jurkat cells via turnover of WST (see example 2). Enzymatic activity was determined as described in example 3. Fractions which show high purity and cytotoxic respectively enzymatic activity (FIG. 1A; fraction 42 to 48) were pooled, concentrated and loaded onto a Superose 12 HR 10/30 column (Pharmacia). Proteins were eluted with 100 mM potassium phosphate buffer (pH 7.2) at a flow rate of 0.5 ml/min The first peak represents the active APIT (FIG. 2B; fraction 11 to 14).

EXAMPLE 2 Phenotype of APIT-Induced Cell Death

The purple fluid of Aplysia punctata contains a cytolytic activity which induces rapid and extensive death of Jurkat T cells in culture. APIT induces cell death of tumor cells which resembles neither apoptosis nor necrosis. In order to classify the APIT-induced cell death we looked for common features of apoptosis and necrosis.

Jurkat T cells were harvested in the log phase, centrifuged and adjusted to a density of 5×10⁵/ml with fresh medium (RPM supplemented with 10% FCS, 100 U/ml penicillin and 100 μg/ml streptomycin). Cells were cultured with APIT, cycloheximide as a positive control or medium at 37° C., 5% CO₂ and 100% humidity for the indicated times. Fragmented DNA of apoptotic cells was analyzed according to Herrmann et al. (1994, Nucleic Acid Research 22: 5506-5507). Cell vitality was determined as metabolic activity via the turnover of WST-1 (ROCHE, Mannheim) to red formazan by the mitochondrial dehydrogenase of viable cells. Absorbance of the cell suspension was measured photometrically at 450 nm (690 nm reference). Toxicity was measured by quantifying propidium iodide uptake (1 μg/ml in PBS) by Flow Cytometry.

Morphologically, tumor cells treated with ink or APIT did not exhibit typical morphological apoptotic or necrotic signs of cell death (FIG. 2A), and neither blebbing nor swollen cells were detected when cells were treated with a lethal dose of ink. Cells did not form clusters anymore, cytoplasm became translucent and nuclei prominent (FIG. 2A). The intracellular movements of plasma and organelles stopped, detachment and formation of vacuoles were observed when adherent cells were incubated with APIT (data not shown). Consistent with the absence of apoptosis, fragmented DNA or nuclei were not detected in ink-treated tumor cells (FIG. 2 B); moreover, caspases were not activated (data not shown). Metabolic activity of tumor cells was blocked as early as 30 min after exposure to ink or APIT (FIG. 2C). Ink-treated tumor cells rapidly took up propidium iodide (PI) indicating plasma membrane permeabilization and cell death (FIG. 2D).

EXAMPLE 3 Stability of APIT

APIT was further characterized by its sensitivity to heat, low pH and high concentrations of urea.

For determination of its heat sensitivity native ink was dialyzed against PBS at 4° C. for several days to separate chromopeptides. Dialysed ink was incubated for 10 min at the indicated temperatures, and activity was measured immediately as enzymatic production of H₂O₂. This assay is based on the finding that APIT transforms L-lysine to H₂O₂ and á-keto acid. The production of H₂O₂ was determined via the turnover of ABTS (2,2-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) to a green formazan in the presence of H₂O₂ by horseradish peroxidase. Heat-treated ink was incubated with L-lysine (1 mM) in 100 Fl 100 mM potassium phosphate buffer, pH 7.2 for 10 min at 25° C. The reaction was stopped by adding 1 Fl of 10 M phosphoric acid. To 25 Fl of this solution 1 mM ABTS and I Unit horseradish peroxidase was added in 225 Fl 100 mM potassium phosphate buffer, pH 5.0. Absorption was measured photometrically at 405 nm (reference 690 nm). Purified APIT was challenged to different pH-values by adding a mixture of monobasic and dibasic potassium phosphate and phoshphoric acid rendering the desired pH. After a 10 min incubation pH of samples was adjusted to pH 7.2 by adding appropriate amounts of dibasic phosphate. Afterwards enzymatic activity was measured as H₂O₂-production as described above.

The activity of APIT after treatment with urea was measured via the production of á-keto acid, which was quantified photometrically by its reaction with the hydrazone MBTH (3-methyl-2-benzothiazolone hydrazone hydrochloride) as described by Soda (1968). Dialyzed ink was incubated with urea at indicated concentrations for 30 min. Subsequently the remaining enzymatic activity was measured without removing urea for 15 min at 25° C. As control, defined amounts of á-keto isocaproic acid (Sigma; K-0629) were treated equally.

APIT was characterized by its heat sensitivity and was found to exhibit a high and constant activity after pre-incubation for 10 min at 0° C. to 50° C. Activity was clearly reduced at 60° C. and absent at temperatures of 70° C. or higher (FIG. 3A). APIT also shows a loss of activity with decreasing pH, with complete inactivation after a 10 min pre-incubation at pH 3 or lower (FIG. 3B). An outstanding feature of APIT is its resistance to urea (FIG. 3C). After 30 min treatment with 6 M urea, the activity of APIT was almost unaffected. At 8 M urea, the activity was reduced by about 50%.

EXAMPLE 4 Sequencing and Cloning of APIT

In order to clone the cDNA of APIT N-terminal and internal peptide sequences were identified by PMF (peptide mass fingerprint), ESI/MS and Edman degradation (FIG. 4A). A suitable internal peptide sequence was used to design a degenerated primer for PCR (FIG. 4A, underlined sequence) with reverse transcribed mRNA, prepared from Aplysia punctata tissues. Subsequent 5′-RACE yielded the full length cDNA which was cloned and analyzed.

Amino Acid Sequencing by Peptide Mass Fingerprint (PMF), ESI/MS and Edman Degradation.

Purified APIT was separated by SDS PAGE and 2 DE gel electrophoresis (Thiede et al., 2001, J. Biol. Chem. 276: 26044-26050). The N-terminus of APIT was identified from a single band/spot of a PVDF blot by Edmann degradation. For the identification of internal peptide sequences a single band/spot was punched from the gel, digested with trypsin and dissolved in aqueous trifluoroacetic acid (Thiede et al., 2001, J. Biol. Chem. 276:26044-26050). Tryptic peptides were separated using a Smart-HPLC system with a column of 2.1 mm inner diameter and 10 cm length (FRPC C2/C18 SC 2.1/10, Smart System, Pharmacia Biotech, Freiburg, Germany) and an acetonitrile gradient in 0.1% (v/v) trifluoroacetic acid at a flow rate of 100 Fl/min at room temperature. The peptide fractions were dried, dissolved in 6 Fl 0.3% (v/v) aqueous trifluoroacetic acid/acetonitrile (2:1) and analyzed by MALDI-MS. The mass spectra were recorded by using a time-of-flight delayed extraction MALDI mass spectrometer (Voyager-Elite, Perseptive Biosystems, Framingham, Mass., USA) as previously described (Thiede et al., 2001, J. Biol. Chem. 276:26044-26050). Briefly, fifty mg/ml 2,5-dihydroxybenzoic acid in 0.3% (v/v) aqueous trifluoroacetic acid/acetonitrile (2:1) was used as matrix and 0.3 Fl of the sample and 0.3 Fl of the matrix were mixed and applied to a gold-plated sample holder and introduced into the mass spectrometer after drying. The spectra were obtained in the reflectron mode by summing 50-150 laser shots. For N-terminal sequencing peptide fractions containing single masses were loaded onto a Biobrene-coated glass fiber filter, transferred to a PVDF membrane and excised. Sequencing was performed using a Procise sequencer (Applied Biosystems, Weiterstadt, Germany).

Cloning of the APIT gene. In order to dissect mantle gland, nidamental gland, digestive gland and opaline gland some animals were relaxized by injection of 5 10 ml sterile MgCl₂ solution (380 mM). Isolated tissues were frozen immediately in liquid nitrogen. Total RNA was prepared from these tissues using the ‘peq gold TRIfast’ reagent (Peqlab). mRNA was reverse transcribed using the tagged oligo dT oligonucleotide 5′-tcc taa cgt agg tct aga cct gtt gca t₍₁₈₎-3′ (SEQ ID NO: 58) (FIG. 4B, oligo 1) and the Superscript II polymerase (LIFE) at 42EC. In order to amplify a fragment of the APIT gene the degenerated primer 5′-tc gtg ttc gar tac tci gay cg-3′ (SEQ ID NO: 59) derived from the APIT peptide VFEYSDR SEQ ID NO: 48) (FIG. 4B, oligo 2) and the specific primer 5′-ctg tag gtc tag acc tgt tgc a-3′ (SEQ ID NO: 60) (FIG. 4B, oligo 3) directed against the tag sequence of the oligo dT-primer was used. PCR was performed with the expand long template system (ROCHE, Mannheim) at 68EC and the product was cloned into the pCMV-vector (Stratgene) and sequenced. The 5′ terminal cDNA of APIT was cloned using the 5′ RACE System (LIFE) according to the manufacturers instructions. Primers 5′-ccg tgt aga tct cac tgc cat a-3′ (SEQ ID NO: 61) (FIG. 4B, oligo 4) or 5′-ccg ttg agt tgt aga cct-3 (SEQ ID NO: 62) (FIG. 4B, oligo 6) were combined with the primers 5′-ggc cac gcg tcg act agt acg ggi igg gii ggg iig-3′ (SEQ ID NO: 63) (FIG. 4B, oligo 5) or 5′-aatt ggc cac gcg tcg act agt ac-3′ (SEQ ID NO: 64) (FIG. 4B, oligo 7) to yield a product which was cloned into the pCDNA3-vector (Invitrogen) and sequenced. Finally, full length APIT cDNA was obtained by amplifying the APIT using the specific primers 5″ aa ttc tcg tct gct gtg ctt ctc ct (SEQ ID NO: 65) (FIG. 4B, oligo 8) and 5″ gac tta gag gaa gta gtc gtt ga (SEQ ID NO: 66) (FIG. 4B, oligo 9) and cloned into the pGEX-4T3 Vector (Amersham). DNA from 3 clones of transfected E. coli was prepared and sequenced.

The identity of the isolated gene was confirmed by comparing the computed translational product (FIG. 4C) with the amino acid sequences of the tryptic peptides (FIG. 4A) and the peptide mass fingerprint. It consisted of 1608 bp coding for a protein of 535 amino acids (FIG. 4C) with the predicted mass of 60,167 dalton and a pI of 4.59. The N-terminal 18 amino acids of APIT comprised a putative secretion signal sequence which was absent from the mature protein, most likely due to posttranslational modification during secretion. Furthermore, APIT exhibited homology to FAD-binding oxidoreductases with a conserved dinucleotide binding fold around amino acids 39 to 66 followed by a so-called GG-motif typical for certain oxidases like LAAO, MAO (FIG. 4C) (Dailey et al., 1998, J. Biol. Chem. 273:13658-13662; Vallon et al., 2000, Proteins 38:95-114; Macheroux et al., 2001 Eur. J. Biochem. 268:1679-1686). The highest degree of homology existed to the Cyplasin from A. punctata, the Aplysianin from A. kurodai and the mucus-toxin of the giant African snail Achatina fulica.

Comparing the 3 derived DNA-sequences we often found differences in the third position of coding triplets which nevertheless only seldom produced changes in the amino acid sequence of APIT (FIG. 4C).

By the method described above, further 11 clones were isolated from Aplysia punctata which have a homology to the sequences described in FIG. 4 of at least 95%. Several mutations of the amino acid sequence were found in the domain comprising the dinucleotide binding fold and the GG motif, which probably have no effect upon the function (FIG. 4D). In Pos. 22 of SEQ ID NO: 2, C is replaced by S in two clones. In Pos. 52, A is replaced by T in one clone. In Pos. 60, L is replaced by Q in 7 clones. In Pos. 69, D is replaced by H in one clone. In Pos. 77, T is replaced by S in one clone.

EXAMPLE 5 FAD Association

The toxic and enzymatic activity of APIT is due to the presence of an attached FAD.

In order to purify the tumor lytic activity, ink from A. punctata was subjected to different purification protocols and afterwards each fraction was tested for its toxic activity (see example 1). Activity always correlated with the presence of a protein of approximately 60 kDa (FIGS. 5 A and B). Moreover, APIT was found to contain carbohydrate residues using the DIG Glycan/Protein double labeling method (Roche; data not shown). Furthermore, all spectra of the highly active fractions exhibited a double peak at 390/470 nm (FIG. 5C) which is characteristic for protein bound flavines (Massey et al., 2000, Biochem Soc. Trans. 28:283-96). Heating of APIT for 10 min to 60° C., which is accompanied by a substantial loss of activity also results in loss of detectable FAD-absorption, as is the case with lowering the pH to inactivating values around pH 3. Heating and pH-challenge of APIT was performed as described in example 3 (data not shown).

Consistently, APIT contained the conserved dinucleotide binding fold involved in pyrophosphate binding (Wierenga et al., 1986, J. Mol. Biol., 187:101-107) which is found in many flavoproteins (FIG. 4B; example 4). Moreover, in APIT like in many oxidases a so-called GG-motif is found adjacent to the dinucleotide binding fold (Dailey et al., 1998, J. Biol. Chem. 273:13658-13662, Vallon et al., 2000, Proteins, 38:95-114). Based on the structure of the dinucleotide binding fold and conserved sequence motifs, FAD containing proteins are ordered into 4 families (Dym et al., 2001, Protein Sci. 10:1712-28). According to this classification and based on homology APIT belongs to the Glutathione reductase 2 family (GR2) (Dym et al., 2001, Protein Sci. 10:1712-28). The data show that FAD is a necessary prosthetic group for toxic and enzymatic activity of APIT.

EXAMPLE 6 Cell-Death is Mediated Via H₂O₂

Proteome analysis revealed that thioredoxin peroxidase II is involved in the APIT mediated tumor cell death. Thioredoxinperoxidase II is involved in detoxification of reactive oxygen species (ROS) by reducing hydrogen peroxides as well as other peroxides. We therefore tested whether H₂O₂ is produced during APIT incubation and found that H₂O₂ is the mediator of APIT-induced cell death. Scavenging this toxic compound by catalase over a certain period of time (6-8 hours) results in survival of APIT treated cells. Notable long-term exposure of tumor cells (>18 hours) with APIT and catalase causes the death of tumor cells by the deprivation of L-lysine and L-arginine. H₂O₂ production was measured after incubation of APIT in medium alone and in cell suspension as described in example 3. Toxicity was measured by quantifying propidium iodide uptake (1 μg/ml in PBS) by Flow Cytometry. Cell vitality was determined as metabolic activity via the turnover of WST-1 (ROCHE, Mannheim) to red formazan by the mitochondrial dehydrogenase of viable cells. Absorbance of the cell suspension was measured photometrically at 450 nm (690 nm reference).

As shown in FIG. 6A, APIT induced the production of H₂O₂ in the presence (167 μM) as well as in absence of cells (280 μM). This strongly argues for an enzymatic activity of APIT which transforms medium ingredients under the production of hydrogen peroxide. In the presence of cells the measured H₂O₂ amount is somewhat lower which might be explained by cellular consumption and degradation of H₂O₂. In the absence of APIT H₂O₂ was not detectable. To investigate whether the APIT-induced cell death is mediated by H₂O₂, cells were treated with APIT in the presence of the H₂O₂ degrading enzyme catalase and then stained with PI. Catalase completely abolished the ink-induced increase of PI stained cells (FIG. 6B). Degradation of H₂O₂ by catalase also inhibited the rapid break-down of metabolic activity induced by APIT (FIG. 6C) but, as expected, was ineffective in blocking CD95 (Fas/Apo-1)-induced cell death in the same assay (FIG. 6C). In the presence of catalase APIT no longer induced morphological changes of tumor cells as judged by microscopic investigation (FIG. 6D). The highly efficient inhibition by catalase in particular suggested that no other substance than H₂O₂ elicits the toxic effect observed in APIT-treated samples. Consistently, H₂O₂ induced the phenotype typical for APIT-treated cells (FIG. 6D). Furthermore, proteome analyses revealed changes in H₂O₂ treated cells which were characteristic of APIT-treated cells. These data together clearly demonstrated that the cytotoxic activity depended on the H₂O₂ producing enzymatic activity of APIT.

Long-term exposure of Jurkat cells to ink from Aplysia punctata in combination with catalase resulted in metabolic activity being decreased to 20% (FIG. 6 E, right panel, white bar). The same result is achieved by treatment with purified APIT in combination with catalase (not shown). Since catalase was effective in inhibiting the H₂O₂-induced loss of metabolic activity completely (FIG. 6E, right panel, black bar), it was concluded that long-term treatment with APIT in the presence of an H₂O₂ scavenger, such as catalase, kills tumor cells not by the remaining low H₂O₂ concentrations but by the deprivation of L-lysine and L-arginine.

EXAMPLE 7 APIT is a L-lysine/L-arginine a-oxidase. Enzymatic Activity is a Prerequisite for Toxicity

APIT produced H₂O₂ in RPMI medium in the absence of cells. In order to identify the substrates in cell culture medium which are converted to H₂O₂ by APIT, we prepared different media with defined amino acid composition by supplementing HEPES buffered modified Krebs Ringer medium (KRG: 25 mM HEPES pH 7.4, 125 mM NaCl, 5 mM KCl, 1.2 mM KH₂PO₄, 5 mM NaHCO₃, 6 mM glucose, 1.2 mM MgSO₄, 1 mM CaCl₂) with 10% FCS, 2 mM glutamine, essential and non-essential amino acids (Invitrogen), or single essential amino acids in concentrations equivalent to RPMI medium (Invitrogen). Media were adjusted to pH 7.4 and filter sterilized. After incubation of these media with purified APIT the enzymatic activity was measured as H₂O₂ production via turnover of ABTS (2,2-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) to a green formazan in the presence of H₂O₂ and horseradish peroxidase (FIG. 7A and Table 1).

In a next step we checked whether the substrate specificity could be impaired by digest of APIT. For proteolytic digest aliquots of dialysed ink were treated for 2 h with proteinase K (0.05 mg/ml final) in PBS at 37° C. Reaction was stopped by adding aprotinin (1 μg/ml final) or PEFA ([4-(2-aminoethyl)-benzolsulfonyl fluoride-hydrochloride]-hydrochloride; 0.25 mg/ml final), and digest was checked on a 15% SDS-PAGE. After incubation of digested ink with different amino acid compositions in potassium phosphate buffer the enzymatic activity was measured as H₂O₂ production (FIG. 7B).

In order to test whether withdrawal of L-lysine and L-arginine results in rescue of APIT-treated cells we incubated Jurkat cells in medium lacking L-lysine and L-arginine. Control cells were cultured in a medium containing L-lysine(HCl (40 mg/l) and L-arginine(HCl (240 mg/l). Toxicity was measured by quantifying propidium iodide uptake (1 μg/ml in PBS) by Flow Cytometry (FIG. 7C).

Cell vitality was determined as metabolic activity via the turnover of WST-1 (ROCHE, Mannheim) to red formazan by the mitochondrial dehydrogenase of viable cells. Absorbance of the cell suspension was measured photometrically at 450 nm (690 nm reference). As control tumor cells were killed by anti-CD95 treatment (FIG. 7D).

á-Keto acids were quantified photometrically by their reaction with the hydrazone MBTH (3-methyl-2-benzothiazolone hydrazone hydrochloride) as described (Soda et al., 1968, Anal. Biochem. 25:228-235) (FIG. 7E).

The K_(m) value for L-lysine was determined as H₂O₂ production and calculated according to Michaelis Menten with the GraphPad Prism 3.0 software (GraphPad Software, San Diego Calif. USA) using non linear regression (FIG. 7F).

Surprisingly, from all amino acids tested only L-lysine and L-arginine served as substrates for APIT to produce hydrogen peroxide (FIG. 7A). Moreover, the restricted substrate specificity was even maintained when APIT was digested with protease K suggesting that the protease resistant fragment of APIT contains both, the active domain and the domain which determines the substrate specificity (FIG. 7B). These data were confirmed by functional analyses which showed that APIT was unable to induce cell death (FIG. 7C) or reduce metabolic activity (FIG. 7D) in tumor cells incubated in medium lacking L-lysine and L-arginine, indicating that the enzymatic activity of APIT is the prerequisite for its toxicity. L-lysine and L-arginine deprivation had no influence on the metabolic activity of tumor cells under the experimental conditions (FIG. 7D). Activation of CD95(Fas/Apo-1) efficiently impaired cell vitality irrespective of the presence of L-lysine or L-arginine (FIG. 7D), demonstrating that cell death can be induced under L-lysine and L-arginine limited conditions.

As shown in the reaction scheme in FIG. 7G, á-keto derivatives are produced by amino acid oxidases and these could indeed be demonstrated when L-lysine was used as substrate for APIT (FIG. 7E). These results suggested that APIT catalyses the formation of H₂O₂ by the reaction outlined in FIG. 7G. Kinetic studies analyzed according to Michaelis-Menten revealed a K_(m) of 0.182 mM for L-lysine (FIG. 7F).

By adding L-lysine (2-50 μg/ml) to tumor cells which are cultured with APIT (20 ng/ml) in medium depleted of L-lysine and L-arginine or in pure FCS, the metabolic activity of the tumor cells can be reduced down to 16% respectively 50% of the control cells without additional L-lysine. This shows that the tumorolytic effect of APIT can be manipulated by changing the amount of available substrate which is of significance for in vivo studies and/or for application of APIT in pharmaceutical compositions and/or methods for treatment of cancer.

EXAMPLE 8 Sensitivity of Different Tumor Cell Lines to APIT Induced Cell Death

Tumor cells were harvested in the log phase. Triplicates of each 50,000 cells were cultured in a flat bottomed 96-well-plate in 100 Fl medium with increasing concentrations of APIT. After 14 hours the metabolic activity of the cells was determined by addition of 10 Fl WST-1 per well (ROCHE, Mannheim). The yellow tetrazolium salt is cleaved to red formazan by cellular enzymes of viable cells. The metabolic activity correlates with cell vitality and was quantified by measuring the absorbance of the dye solution with a spectrophotometer at 450 nm (reference 650 nm).

APIT is able to kill different tumor cells. T and B cell leukemia cell lines Jurkat neo, CEM neo, SKW neo), a chronic myelogenous leukemia cell line (1K562), and cells from an orphan and aggressive osteosarcoma (Ewings tumor: RDES, A673) showed the highest sensitivity to the APIT induced cell death IC₅₀≦5.6 ng/ml), followed by cells derived from small cell lung cancer (GLC4, GLC4/ADR), cervix cancer (Chang) and acute monocytic leukemia (THP-1) IC₅₀<10 ng/ml). Most of the adherent growing cells of solid tumors (breast cancer: MCF-7, SK-BR-3; prostate cancer: PC3, DU-145; colon cancer: HT-29; cervix cancer: HeLa; uterus cancer: Hec-1-B; larynx cancer HEp-2; stomach cancer: AGS; liver cancer: Hep G2) and the monocyte leukemia cell line MonoMac 6 are less sensitive at the indicated cell concentration IC₅₀<20 ng/ml), but become more sensitive when lower cell concentrations were used IC₅₀ 5-10 ng/ml).

Resistance to apoptosis as well as multi drug resistance (MDR) represent severe problems in cancer therapy. It is therefore of particular interest that APIT kills apoptosis resistant cell lines as well as MDR cancer cell lines equally efficient as their non resistant counter parts (Tab. 2): Over-expression of apoptosis inhibitors of the Bcl-2 family in acute lymphoblastic leukemia cell lines (CEM Bcl-X_(L), Jurkat Bcl-2) as well as in B cell leukemia (SKW Bcl-2) (Tab. 2; 4th row) does not protect from APIT mediated cell death and results in IC₅₀ values of <6 ng/ml, similar to the non-transfected parental cell lines, confirming that APIT induce cell death in an apoptosis independent way. The MDR cell line GLC4/ADR (Tab. 2,5th row) was generated by selection with doxorubicin (Zijlstra et al., 1987, Cancer Res. 47:1780-1784). Its multifactorial MDR is caused by over-expression of MRP-1 and a decreased activity of the DNA topoisomerase II. GLC4/ADR cells possess almost the same sensitivity to APIT (IC₅₀ 10 ng/ml) as the parental line GLC4 does IC₅₀ 9 ng/ml).

EXAMPLE 9 Proteome Analysis: Change in Protein Expression Pattern in Jurkat T Cells after Treatment with APIT

Treatment with APIT. Jurkat T cells (5×10⁵/ml) were incubated with APIT (20 ng/ml) for 8 h at 37° C. in 5.0% CO₂ in the presence of 1 μg/ml cycloheximide. Controls were performed without APIT.

Total cell lysate. The Jurkat T cells were solubilized in 5 volumes of a buffer containing 9 M urea, 25 mM Tris/HCl, pH 7.1, 50 mM KCl, 3 mM EDTA, 70 mM DTT, 2.9 mM benzamidine, 2.1 μM leupeptin, 0.1 μM pepstatin, 1 mM PMSF, and 2% carrier ampholytes (Servalyte pH 2-4, Serva, Heidelberg, Germany). After 30 minutes of gentle stirring at room temperature, the samples were centrifuged at 100000 g (Ultracentrifuge Optima TLX, Beckman, München, Germany) for 30 minutes with a TLA120.2 rotor, which were kept at room temperature before centrifugation. The clear supernatant was frozen at −70° C.

Proteomics. The methods of preparing 2-DE gels, staining with Coomassie Blue G-250, staining with silver nitrate, in-gel tryptic digestion, peptide mass fingerprinting by MALDI-MS, and identification of the proteins are described in Jungblut et al., Molecular Microbiology, 2000, 36, 710-725.

Identification was performed using the peptide mass fingerprinting analysis software MS-Fit (available via the world wide web at prospector.ucsf.edu/ucsfhtml3.2/msfit.htm) or ProFound (available via the world wide web at canada.proteometrics.com/prowl-cgi/ProFound.exe?FORM=1). Searches were performed in the databases NCBInr and SwissProt. The proteins are referenced by the genbank identifier, accession number and/or version number.

Results. APIT induces either upregulation, downregulation, or modification of the proteins. Modification in the context of this example is a change in the apparent mass and/or the apparent pI value of the protein. By comparison of 2-DE patterns of APIT-treated whole cell lysates with the corresponding patterns of untreated cells, the proteins as described in Table 3 were identified to be affected by APIT.

EXAMPLE 10 Transcriptome Analysis

The influence of APIT on the gene expression of tumor cells was investigated by Microarray technology.

In situ Oligonucleotide Arrays. A custom oligonucleotide glass array of specific 60 mer oligonucleotides representing the mRNA of about 8500 human genes was designed based on human Unigene clusters (Unigene build No. 148) including positive and negative control oligonucleotides (Homo sapiens house keeping genes and Arabadopsis thaliana genes respectively). The probe design included a base composition filter and a homology search to minimise cross-hybridisation.

RNA isolation, labelling and hybridisation to arrays. Jurkat neo cells (1×10⁷ in 20 ml) were cultured for 2 hours in medium (RPMI+10% FCS) in the presence or absence of APIT (10 ng/ml) at 37° C., 5% CO₂. Cells were harvested and the pellet was dissolved in 2 ml Trizol (Life Technologies). Total RNA was extracted after addition of chloroform and subsequent centrifugation and precipitated with isopropanol. After washing the pellet with 75% ethanol it was briefly air-dryed. Quality control of the RNA included exclusion of genomic DNA by PCR and “Lab on a chip technology” (Bioanalyser). RNA (5 μg) from each pool was amplified using a reverse transcriptase/T7 polymerase. 1.5 μg of test cRNAs labelled either with Cy3 or Cy5 were hybridised for 16 hours at 65° C. to arrays. Each sample was also labelled and hybridised with the reverse fluorophore to obviate possible dye bias. Slides were scanned using a Microarray scanner. Background signal was determined using negative control spots and subtracted, data were normalised relative to non-regulated genes. Data from duplicate hybridizations were combined.

Results. Tables 4 and 5 summarize the genes with increased or decreased transcription rate of treated cells compared with untreated cells, indicating these genes and/or its gene products (proteins) to be targets of APIT and/or H₂O₂.

EXAMPLE 11 Knock Down of Prx I Sensitized Tumor Cells for APIT Induced Cell Death

Peroxiredoxin I (Prx I) exhibited the most significant modification observed in 2-DE protein patterns of APIT treated cells in comparison to untreated Jurkat cells (Table 3). The modification of Prx I which is observed in 2-DE gel analysis of APIT treated cells resembles that described for the oxidized and inactivated Prx I, indicating that APIT inactivates this detoxification system. In order to investigate the role of Prx I for the APIT induced cell death we performed knock down of Prx I expression by RNA interference (RNAi). If Prx I was involved in the detoxification of H₂O₂ produced by APIT, we expected to observe a sensitization in cells in which Prx I expression is decreased. Therefore, 20.000 HeLa cells/well were seeded in a 96 well plate one day prior to transfection. Transfection was performed with 0.25 μg siRNA directed against

Prx I having the sequence (SEQ ID NO: 9):

5′-GGCUGAUGAAGGCAUCUCGdTdT-3′ (SEQ ID NO: 73) 3′-dTdTCCGACUACUUCCGUAGAGC-5′, Lamin A/C having the sequence (SEQ ID NO: 30):

5′-CUGGACUUCCAGAAGAACAdTdT (SEQ ID NO: 74) 3′-dTdTGACCUGAAGGUCUUCUUGU-5′, and Luciferase having the sequence (SEQ ID NO: 31):

5′-CUUACGCUGAGUACUUCGAdTdT-3′ (SEQ ID NO: 75) 3′-dTdTGAAUGCGACUCAUGAAGCU-5′, as control and 2 il transmessenger per well using the transmessenger transfection kit (Qiagen, Hilden, Germany) according to manufacturers instructions. For APIT treatment (40 ng/ml) transfections were conducted in triplicates. 24 h after transfection cells were splitted and grown for additional 48 h before fresh medium with or without APIT was added for 6 h. Assay conditions which led to a 50 to 70% reduction of the metabolic activity of treated cells were chosen for RNAi experiments. Metabolic activity was determined as described in Example 2. In parallel, RNA from about 50.000 cells was isolated using the RNeasy 96 BioRobot 8000 system (Qiagen) 48 h after transfection. The relative amount of mRNA was determined by realtime PCR using Quantitect™ SYBR Green RT-PCR Kit from Qiagen following manufacturers instructions. The expression level of Prx mRNA was normalised against the internal standard GAPDH. The following primers were used: Prx I 5′: CTGTTATGCCAGATGGTCAG (SEQ ID NO: 67), Prx I 3′: GATACCAAAGGAATGTTCATG (SEQ ID NO: 68), Lamin A/C5′:CAAGAAGGAGGGTGACCTGA (SEQ ID NO: 69), Lamin A/C3′:GCATCTCATCCTGAAGTTGCTT (SEQ ID NO: 70), GAPDH 5′:GGTATCGTGGAAGGACTCATGAC (SEQ ID NO: 71), GAPDH 3′:ATGCCAGTGAGCTTCCCGTTCAG (SEQ ID NO: 72).

To measure sensitization, conditions were chosen under which the reduction of metabolic activity of treated cells was 50% or less of the untreated cells. siRNAs were transfected into HeLa cells and after 72 h cells were treated with APIT for 6 h and metabolic activity was determined. In parallel, cells were harvested for quantitative analysis of the respective mRNAs by realtime PCR (FIG. 8 A). The mRNA of Prx I was reduced by more than 90% compared to the mRNA level of GAPDH. Interestingly, this reduction of Prx I expression significantly sensitized the cells for killing by APIT whereas control siRNA directed against Luciferase and Lamin A/C had no effect (FIG. 8 B). Our data show that knock down of Prx I by RNAi rendered the cells hypersensitive for APIT suggesting that Prx I is part of an H₂O₂ detoxifying pathway which is modulated by APIT.

In summary, we identified the modification of Prx I, as an important step in the APIT of this detoxification system. The fact that the knock down of Prx I expression by RNAi increased the sensitivity of tumor cells for the cytolytic activity of APIT underlines the impact of Prx I RNA interference for cancer therapy.

EXAMPLE 12 APIT Does Not Induce Actin Depolymerisation

Cyplasin S and L, proteins from Aplysia punctata which induce cell death of tumor cells were described to cause fast actin depolymerisation in human tumor cells (see WO 03/057726). The influence of APIT treatment on actin filaments by fluorescence staining of actin by Phalloidin-TRITC (Tetramethylrhodamin-isothiocyanat) is investigated.

HeLa cells (1.5×105 cells/well/ml) were cultured over night on cover slips in 12 well plates. Subsequently, cells were incubated in the presence or absence of APIT (40 ng/ml) for 6 h or Cytochalasin D (1 ìM; Sigma 8273) for 30 min. After washing in PBS, cells on cover slips were fixed for 10 min in 3.7% PFA (paraformaldehyde), washed again and finally permeabilized by a 1 min incubation in 0.5% Triton X-100. Blocking of unspecific binding sites by incubation in PBS, 1% FCS, 0.05% Tween 20 was followed by actin staining with Phalloidin-TRITC in blocking puffer for 15 min and 3 fold washing. Nuclei were stained by the presence of Hoechst 33258 in the last washing step. Cover slips were investigated by fluorescence microscopy.

As shown in FIG. 9 untreated cells (A) possess a typical actin cytoskeleton. Incubation in the presence of Cytochalasin (B), an inducer of rapid actin depolymerisation, resulted in a massive loss of actin filaments and an accumulation of actin in clumps. In contrast, APIT(C) did not induce actin depolymerisation in HeLa cells. APIT treated cells remain their actin filaments, even after 6 h when the plasma membrane was already disrupted (see example 2, FIG. 2D). This clearly differentiates APIT induced cell death from that induced by Cyplasins.

EXAMPLE 13 Healthy Human Cells are Resistant Against the APIT-Induced Cell Death

To analyze the specificity of APIT for tumor cells, normal human umbilical vein endothelial cells (HUVEC) and tumor cells (Jurkat cells) were incubated with increasing amounts of purified APIT and analyzed for lactate dehydrogenase (LDH) release (FIG. 10).

HUVEC and Jurkat cells (50,000 cells/100 ìl/wells) were treated with increasing amounts of APIT in a 96 well plate. After over night incubation half of the culture supernatants (50 ìl) were transferred in fresh wells and mixed with 50ìl reagent of Cytotoxicity Detection Kit-LDH according to the manufacturers instruction (Roche 1644793). Release of LDH in the supernatant is found only, when cells were killed by APIT. LDH release was calculated as the ratio of LDH activity of APIT treated cells relative to the LDH activity of Triton X 100 lysed cells.

Jurkat cells showed a dramatic release of LDH upon incubation with APIT (FIG. 10). In contrast, even at the highest APIT concentrations used in this experiments (40 ng/ml), APIT treated HUVEC cells only showed a minor LDH release below 10%, indicating a strong resistance of these normal cells against the cytolytic activity of APIT. As several tumor cell lines showed a similar APIT sensitivity as the Jurkat cells (Table 2), the data suggest the toxic effect induced by APIT is tumor specific.

TABLE 1 EAA (essential amino acids) L-arainine•HCl 126.4 mg/l L-cystine 24.02 mg/l L-histidine•HCl•H2O 41.92 mg/l L-isoleucine 52.46 mg/l L-leucine 52.46 mg/l L-lysine•HCl 73.06 mg/l L-methionine 14.92 mg/l L-phenylalanine 33.02 mg/l L-threonine 47.64 mg/l L-tryptophane 10.2 mg/l L-tyrosine 36.22 mg/l L-valine 46.86 mg/l NEAA (non-essential aino acids) L-alanine 8.9 mg/l L-asparagine 13.2 mg/l L-aspartic-acid 13.3 mg/l L-alutamic acid 14.7 mg/l glycine 7.5 mg/l L-prolin 11.5 mg/l L-serine 10.5 mg/l Single amino acids histidine•HCl•H2O 20 mg/l isoleucine 50 mg/l leucine 50 mg/l methionine 15 mg/l phenylalanine 15 mg/l threonine 20 mg/l tyrosine 20 mg/l arginine•HCl 240 mg/l lysine 40 mg/l D-lysine 40 mg/l cystine 50 mg/l tryptophane 5 mg/l valine 20 mg/l glutamine 300 mg/l

TABLE 2 APIT kills different tumor cell lines IC50 models for kind of tumor tumor cell line (ng/ml) 1. solid lung cancer GLC4 9 tumors breast cancer MCF-7, SK-BR-3 * prostate cancer PC3, DU145 * colon cancer HT-29 20 cervix cancer HeLa, Chang *, 10 uterus carcinoma Hec-1-B * larynx cancer HEp-2 * stomach cancer AGS * liver cancer Hep G2 * 2. leukemia T cell leukemia (ALL) Jurkat neo 3.2 T cell leukemia (ALL) CEM neo 5.6 B cell leukemia SKW neo 3 Monocyte leukemia (AML) Mono Mac 6 * Monocyte leukemia (AML) THP-1 10 3. “orphan” Ewings sarcoma RDES 4.5 tumors A673 5 4. apoptosis (CML) K562 4.25 resistant T cell leukemia (ALL) Jurkat Bcl-2 2.7 tumors T cell leukemia (ALL) CEM Bcl-X_(L) 4.0 B cell leukemia SKW Bcl-2 5.5 5. MDR Lung cancer GLC4-ADR 10 tumors

TABLE 3 Proteome analysis Description gi NCBI NCBI version swissprot effect Aldolase A (E.C.4.1.2.13) 229674 1ALD 1ALD P04075 26S proteasome regulatory chain 12 2134660 S65491 S65491 — − 3-Hydroxyacyl-CoA dehydrogenase 2078327 AAB54008 AAB54008.1 Q16836 − C-1-tetrahydrofolate synthase, cytoplasmic (C1-THF synthase) 115206 P11586 P11586 P11586 − Chain A, Structure Of Human Glutamate Dehydrogenase-Apo Form 20151189 1L1F_A 1L1F_A — m or Glutamate dehydrogenase 1 4885281 NP_005262 NP_005262.1 P00367 Cleavage and polyadenylation specific factor 5, 25 kD subunit 5901926 NP_008937 NP_008937.1 + Cofilin 1 5031635 NP_005498 NP_005498.1 P23528 − Coronin, actin binding protein, 1A 5902134 NP_009005 NP_009005.1 P31146 + Dihydrolipoamide dehydrogenase precursor; E3 component of pyruvate 4557525 NP_000099 NP_000099.1 P09822 − dehydrogenase dJ553F4.4 (Novel protein similar to Drosophila CG8055 protein) 12314022 CAC14088 CAC14088.1 + DNA replication licensing factor MCM4 1705520 P33991 + Elongation factor1-delta (EF-1-delta) 20141357 P29692 P29692 P29692 − Enolase 1, alpha; phosphopyruvate hydratase 4503571 NP_001419 NP_001419.1 Q05524 + Glyceraldehyde-3-phosphate dehydrogenase 31645 CAA25833 CAA25833.1 P04406* + or uracil DNAglycosylase 35053 CAA37794 CAA37794.1 P04406* Heat shock 60 kD protein 1 (chaperonin) 14603309 AAH10112 AAH10112.1 Q96FZ6 − Heat shock 60 kDa protein 1 (chaperonin) 4504521 NP_002147 NP_002147.1 P10809 − Heat shock 70 kD protein 9B (mortalin-2) 4756570 NP_004125 NP_004125.1 Q8N1C8 − Heterogeneous nuclear ribonucleoprotein C, isoform b 4758544 NP_004491 NP_004491.1 P07910 m Hspc117 6841456 AAF29081 AAF29081.1 Q9P037 m Inosine-5′-monophosphate dehydrogenase 2 (IMP dehydrogenase 2) 124419 P12268 P12268 P12268 + Isocitrate dehydrogenase 3 (NAD+) alpha 5031777 NP_005521 NP_005521.1 P50213 − KH-type splicing regulatory protein (FUSE binding protein 2) 4504865 NP_003676 NP_003676.1 — − Nuclear matrix protein NMP200 related to splicing factor PRP19 7657381 NP_055317 NP_055317.1 Q9UMS4 − Nucleobindin 2 4826870 NP_005004 NP_005004.1 P80303 − 54 kDa nuclear RNA- and DNA-binding protein (p54(nrb)) (p54nrb) 13124797 Q15233 Q15233 Q15233 + Peroxiredoxin 1 (Thioredoxin peroxidase 2) 548453 Q06830 Q06830 Q06830 m Peroxiredoxin 1; Proliferation-associated gene A; proliferation-associated 4505591 NP_002565.1 NP_002565.1 Q06830 m gene A Peroxiredoxin 2 (Thioredoxin peroxidase 1) 2507169 P32119 P32119 P32119 + Peroxiredoxin 3; antioxidant protein 1; thioredoxin-dependent peroxide 5802974 NP_006784 NP_006784.1 P30048 − reductase precursor 2-phosphopyruvate-hydratase alpha-enolase; carbonate dehydratase 693933 CAA59331 CAA59331.1 P06733 + Proteasome subunit alpha type 7 12643540 O14818 O14818 O14818 + Proteasome subunit beta type 1 (Proteasome component G5) (Macropain 130853 P20618 P20618 P20618 + subunit C5) Ras-GTPase-activating protein SH3-domain-binding protein; GAP binding 5031703 NP_005745.1 NP_005745.1 Q13283 m protein Replication protein A2, 32 kDa 4506585 NP_002937 NP_002937.1 P15927 − Rho GDP-dissociation inhibitor 2 (Rho GDI 2) (Rho-GDI beta) (Ly-GDI) 1707893 P52586 P52566 P52566 − Ribosomal protein P0; 60S acidic ribosomal protein P0 4506667 NP_000993 NP_00993.1 P05388 − or similar BLOCK 23 20536934 XP_165448 XP_165448.1 Q8NHW5 Ribosomal protein, large, P0 12654583 AAH01127 AAH01127.1 P05388 − RNA-binding protein regulatory subunit 6005749 NP_009193 NP_009193.1 O14805 + RNA-binding protein regulatory subunit 12720028 XP_001707 XP_001707.2 O14805 + Semenogelin I; Semenogelin 4506883 NP_002998 NP_002998.1 P04279 − Similar to villin 2 (ezrin) 15530243 AAH13903 AAH13903.1 P15311 − Splicing factor proline/glutamine rich (polypyrimidine tract binding protein 4826998 NP_005057 NP_005057.1 P23246 − associated) Stathmin 1; metablastin; prosolin; oncoprotein 18; phosphoprotein 19; 5031851 NP_005554 NP_005554.1 − leukemia-associated phosphoprotein p18 U2 small nuclear ribonucleoprotein A′ (UZ snRNP-A′) 134094 P09661 P09661 P09661 + Vimentin 4507895 NP_003371 NP_003371.1 P08670 − Voltage-dependent anion-selective channel protein 2 (VDAC-2) (hVDAC2) 1172554 P45880 P45880 P45880 −

TABLE 4 Transcriptome analysis Unigene cluster Description GENE gi pir/NCBI/swisspr

effect Hs.3833 3′-phosphoadenosine 5′-phosphosulfate synthase 1 PAPSS1 4885537 NP_005434.1 − Hs.166563 replication factor C (activator 1) 1, 145 kDa RFC1 15011931 ref:NP_002904.2 − Hs.78991 DNA segment, numerous copies, expressed probes (GS1 gene) DXF68S1E 6912346 ref:NP_036212.1 − Hs.326035 early growth response 1 EGR1 119242 sp:P18146 ++ Hs.108885 collagen, type VI, alpha 1 COL6A1 15011913 ref:NP_001839.1 ++ Hs.78944 regulator of G-protein signalling 2, 24 kDa RGS2 2135146 pir:I53020 ++ Hs.110571 growth arrest and DNA-damage-inducible, beta GADD45B 9945332 ref:NP_056490.1 ++ Hs.78465 v-jun sarcoma virus 17 oncogene homolog (avian) JUN 135298 sp:P05412 + Hs.82646 DnaJ (Hsp40) homolog, subfmally B, member 1 DNAJB1 1706473 sp:P25685 + Hs.169840 TTK protein kinase TTK 346403 pir:A42861 + Hs.211601 mitogen-activated protein kinase kinase kinase 12 MAP3K12 18202489 sp:Q12852 + Hs.345728 suppressor of cytokine signaling 3 SSI-3 4507235 ref:NP_003946.1 + Hs.3776 zinc finger protein 216 ZNF216 5174755 ref:NP_005998.1 + Hs.73037 cannabinoid receptor 2 (macrophage) CNR2 450068 prf:1920360A + Hs.167578 EST, FLJ25357 hypothetical protein FLJ25357 740170 2004399A + Hs.8715 hypothetical protein MGC3232 MGC3232 3024681 sp:O00268 + Hs.74520 spinocerebellar ataxia 1 (olivopontocerebellar ataxia 1, SCA1 1082237 pir:S46268 + autosomal dominant, ataxin 1) Hs.6151 pumilio homolog 2 (Drosophila) PUM2 14277945 pdb:1IB3 + Hs.8026 EST, Highly similar to SES2_HUMAN Sestrin 2 [H. sapiens] 13633882 sp:P58004 + Hs.82173 TGFB inducible early growth response TIEG 11387050 sp:Q13118 + Hs.198307 von Hippel-Lindau binding protein 1 VBP1 4507873 ref:NP_003363.1 + Hs.179982 tumor protein p53-binding protein TP53BPL 5032191 ref:NP_005793.1 + Hs.2549 adrenergic, beta-3-, receptor ADRB3 1070630 pir:QRHUBE + Hs.2128 dual specificity phosphatase 5 DUSP5 12707566 ref:NP_004410.2 + Hs.36927 heat shock 105 kD HSP105B 5729879 ref:NP_006635.1 + Hs.77558 high mobility group nucleosomal binding domain 3 HMGN3 2495254 sp:Q15651 + Hs.460 activating transcription factor 3 ATF3 88875 pir:C34223 + Hs.104125 adenylyl cyclase-associated protein CAP 399184 sp:Q01518 + Hs.24719 modulator of apoptosis 1 MAP-1 11545896 ref:NP_071434.1 + Hs.8257 cytokine inducible SH2-containing protein CISH 13124022 sp:Q9NSE2 + Hs.101383 ESTs, Weakly similar to A43932 mucin 2 precursor, intestinal - 2135765 pir:A43932 + human (fragments) Hs.276770 CDW52 antigen (CAMPATH-1 antigen) CDW52 4502761 ref:NP_001794.1 + Hs.8084 hypothetical protein dJ465N24.2.1 DJ465N24.2.1 10092679 ref:NP_064713.1 + Hs.78829 ubiquitin specific protease 10 USP10 11360280 pir:T47164 + Hs.889 Charot-Leyden crystal protein CLC 1942831 pdb:1LCL + Hs.277401 bromodomain adjacent to zinc finger domain, 2A BAZ2A 7304921 ref:NP_038477.1 + Hs.300863 lethal (3) malignant brain tumor l(3)mbt protein (Drosophila) homolog H-L(3)M8T 14141728 ref:NP_056293.2 + Hs.4552 ubiquilin 2 UBQLN2 16753207 ref:NP_038472.2 + Hs.151963 GrpE-like protein cochaperone HMGE 18202951 sp:Q9HAV7 + Hs.36606 EST, Weakly similar to T29982 hypothetical protein F11G11.12 - + [C. elegans] HS.85302 adenosine deaminase, RNA-specific, B1 (RED1 homolog rat) ADARB1 2829669 sp:P78563 + Hs.113823 ClpX caseinotytic protease X homolog (E. coli) CLPX 14916956 sp:O76031 + Hs.25911 HLA-B associated transcript 2 BAT2 18375626 ref:NP_542417.1 + Hs.95821 osteoclast stimulating factor 1 OSTF1 11134088 sp:Q92882 + Hs.11217 KIAA0877 protein KIAA0877 + Hs.301064 arfaptin 1 HSU52521 1703203 sp:P53367 + Hs.276238 EST, Moderately similar to kinase suppressor of ras [(Mus musculus] + Hs.211569 G protein-coupled receptor kinase 5 GPRK5 2135145 pir:A48277 + Hs.25524 protein tyrosine phosphatase, non-receptor type 23 PTPN23 7512735 pir:T14756 + Hs.94498 leukocyte immunoglobulin-like receptor, subfamily A (with TM domain), LILRA2 5803068 ref:NP_006857.1 + member 2 Hs.24427 DKFZP566O1646 protein DC8 7512839 pir:T08737 + Hs.46 platelet-activating factor receptor PTAFR 107346 pir:A40191 + Hs.90800 EST, Highly similar to matrix metalloproteinase 16, isoform 1; 13027802 ref:NP_005932.2 + membrane-type matrix metalloproteinase 3; membrane-type-3 matrix metalloproteinase [Homo sapiens] Hs.81648 hypothetical protein FLJ11021 similar to splicing factor, arginine/ FLJ11021 2833266 sp:Q15696 + serine-rich 4 Hs.80338 Bcl-2-associated transcription factor BTF 7661958 ref:NP_055554.1 + Hs.238407 EST, Weakly similar to hypothetical protein FLJ20489 [Homo sapiens] 8923452 ref:NP_060312.1 + [H. sapiens] Hs.154668 KIAA0391 gene product KIAA0391 3024899 sp:O15091 + Hs.76666 chromosome 9 open reading frame 10 C9orf10 13431358 sp:Q9NZB2 + Hs.9701 growth arrest and DNA-damage-inducible, gamma GADD45G 5729836 ref:NP_006696.1 + Hs.100527 connector enhancer of KSR2 CNK2 7662368 ref:NP_055742.1 + Hs.77274 plasminogen activator, urokinase PLAU 224665 prf:1110198A + Hs.93516 ESTs + Hs.376709 Homo sapiens cDNA FLJ33768 fis, clone BRHIP2000021 + Hs.110299 mitogen-activated protein kinase kinase 7 MAP2K7 4826946 ref:NP_005034.1 + Hs.31396 ESTs, Weakly similar to S28807 collagen alpha 1(X) chain precursor + [M. musculus] Hs.129715 gonadatropin-releasing hormone 2 GNRH2 3913735 sp:O43555 + Hs.169370 FYN oncogene related to SRC, FGR, YES FYN 125370 sp:P06241 + Hs.82007 methionyl aminopeptidase 1 METAP1 1703270 sp:P53582 + Hs.239018 RAB11B, member RAS oncogene family RA811B 1082426 pir:JC2487 + Hs.126852 solute carrier family 6 (neurotransmitter transporter, GABA), member 13 SLC6A13 7705539 ref:NP_057699.1 +

indicates data missing or illegible when filed

TABLE 5 Transcriptome analysis GENBANK GENENAME SYMBOL EFFECT NM_005252 v-fos FBJ murine osteosarcoma viral oncogene homolog FOS ++ NM_006705 growth arrest and DNA-damage-inducible, gamma GADD45G * ++ NM_001964 early growth response 1 EGR1 * ++ NM_002228 v-jun sarcoma virus 17 oncogene homolog (avian) JUN * ++ NM_015675 growth arrest and DNA-damage-inducible, beta GADD45B * ++ NM_001124 adrenomedullin ADM ++ NM_005346 heat shock 70 kDa protein 1B HSPA1B ++ NM_002166 inhibitor of DNA binding 2, dominant negative helix-loop-helix protein ID2 ++ NM_004417 dual specificity phosphatase 1 DUSP1 ++ NM_003745 suppressor of cytokine signaling 1 SOCS1 ++ NM_002923 regulator of G-protein signalling 2, 24 kDa RGS2 * ++ NM_005627 serum/glucocorticoid regulated kinase SGK ++ BC012321 activity-regulated cytoskeleton-associated protein ARC ++ NM_025195 phosphoprotein regulated by mitogenic pathways C8FW + NM_030751 transcription factor 8 (represses interleukln 2 expression) TCF8 + NM_014330 protein phosphatase 1, regulatory (inhibitor) subunit 15A PPP1R15A + NM_004083 DNA-damage-inducible transcript 3 DDIT3 + NM_001841 cannabinoid receptor 2 (macrophage) CNR2 * + NM_004024 activating transcription factor 3 ATF3 * + NM_001706 B-cell CLL/lymphoma 6 (zinc finger protein 51) BCL6 + NM_004428 ephrin-A1 EFNA1 * + NM_004419 dual specificity phosphatase 5 DUSP5 * + NM_003088 fascin homolog 1, actin-bundling protein (Strongylocentrotus purpuratus) FSCN1 + AB014566 dishevelled associated activator of morphogenesis 1 DAAM1 + NM_006145 DnaJ (Hsp40) homolog, subfmaily B, member 1 DNAJB1 * + NM_004962 growth differentiation factor 10 GDF10 + D79994 kidney ankyrin repeat-containing protein KANK + NM_006301 mitogen-activated protein kinase kinase kinase 12 MAP3K12 * + NM_002928 regulator of G-protein signalling 16 RGS16 + NM_003955 suppressor of cytokine signaling 3 SOCS3 + NM_004430 early growth response 3 EGR3 + NM_001731 B-cell translocation gene 1, anti-proliferative BTG1 + NM_012342 putative transmembrane protein NMA + NM_002262 killer cell lectin-like receptor subfamily D, member 1 KLRD1 + NM_006007 zinc finger protein 216 ZNF216 * + NM_000905 neuropeptide Y NPY + NM_004418 dual specificity phosphatase 2 DUSP2 + NM_031459 sestrin 2 SES2 + AF332558 BCL2 binding component 3 BBC3 + NM_006000 tubulin, alpha 1 (testis specific) TUBA1 + NM_006644 heat shock 105 kDa/110 kDa protein 1 HSPH1 + L24498 growth arrest and DNA-damage-inducible, alpha GADD45A + AK024029 modulator of apoptosis 1 MOAP1 + NM_005409 chemokine (C-X-C motif) ligand 11 CXCL11 + NM_003383 very low density lipoprotein receptor VLDLR + AF267856 hypothetical protein dJ465N24.2.1 DJ465N24.2.1 * + NM_002450 metallothionein 1L MT1L + NM_001828 Charot-Leyden crystal protein CLC * + NM_013370 pregnancy-induced growth inhibitor OKL38 + AB014581 l(3)mbt-like (Drosophila) L3MBTL + NM_0008875 pim-2 oncogene PIM2 + AL031695 actin, gamma pseudogene 3 AGTGP3 + AI985514 ribosomal protein S19 RPS19 + NM_080686 HLA-B associated transcript 2 BAT2 + NM_021184 chromosome 6 open reading frame 47 C6orf47 + NM_015471 DKFZP566O1646 protein DCS * + NM_000952 platelet-activating factor receptor PTAFR + BC012625 protein phosphatase 1, regulatory (inhibitor) subunit 3C PPP1R3C + NM_023012 hypothetical protein FLJ11021 similar to splicing factor, arginine/serine-rich 4 FLJ11021 * + AK024358 macrophage expressed gene 1 LOC219972 + NM_002658 plasminogen activator, urokinase PLAU * + U12767 nuclear receptor subfamily 4, group A, member 3 NR4A3 + NM_016615 solute carrier family 6 (neurotransmitter transporter, GABA), member 13 SLC6A13 + NM_002135 nuclear receptor subfamily 4, group A, member 1 NR4A1 + AJ251595 CD44 antigen (homing function and Indian blood group system) CD44 − NM_005433 v-yes-1 Yamaguchi sarcoma viral oncogene homolog 1 YES1 − NM_006325 RAN, member RAS oncogene family RAN − NM_004775 UDP-Gal:betaGlcNAc beta 1,4-galactosyltransferase, polypeptide 6 B4GALT6 − AK056671 upstream regulatory element binding protein 1 UREB1 − NM_022817 period homolog 2 (Drosophila) PER2 − L07044 calcium/calmodullin-dependent protein kinase (CaM kinase) II gamma CAMK2G − NM_014890 downregulated in ovarian cancer 1 DOC1 − NM_001782 CD72 antigen CD72 − NM_005766 FERM, RhoGEF (ARHGEF) and pleckstrin domain protein 1 (chondrocyte-derived) FARP1 − NM_000566 Fc fragment of IgG, high affinity Ia, receptor for (CD64) FCGR1A − NM_003036 v-ski sarcoma viral oncogene homolog (avian) SKI − NM_001713 betaine-homocysteine methyltransferase BHMT − NM_001682 ATPase, Ca++ transporting, plasma membrane 1 ATP2B1 − NM_003985 tyrosine kinase, non-receptor, 1 TNK1 − NM_004752 glial cells missing homolog 2 (Drosophila) GCM2 − BC001619 aldehyde dehydrogenase 1 family, member B1 ALDH1B1 − NM_002422 matrix metalloproteinase 3 (stromelysin 1, progelatinase) MMP3 − NM_003024 intersectin 1 (SH3 domain protein) ITSN1 − NM_002613 3-phosphoinositide dependent protein kinase-1 PDPK1 − NM_000098 carnitine palmitoyltransferase II CPT2 − BC002712 v-myc myelocytomatosis viral related oncogene, neuroblastoma derived (avian) MYCN − NM_003112 Sp4 transcription factor SP4 − NM_012062 dynamin 1-like DNM1L − NM_000880 interleukin 7 IL7 − NM_004564 PET112-like (yeast) PET112L − NM_001771 CD22 antigen CD22 − AA904067 protein phosphatase 1, regulatory (inhibitor) subunit 12B PPP1R12B − NM_001633 alpha-1-microglobulin/bikunin precursor AMBP − NM_007216 Hermansky-Pudlak syndrome 5 HPS5 − AV708310 protein phosphatase 2 (formerly 2A), catalytic subunit, alpha isoform PPP2CA − AF296765 cerebral cavernous malformations 1 CCM1 − AF155117 kinesin family member 21A KIF21A − NM_002006 fibroblast growth factor 2 (basic) FGF2 − NM_004362 calmegin CLGN − NM_021221 lymphocyte antigen 6 complex, locus G5B LY6G5B − AK001541 secretory carrier membrane protein 1 SCAMP1 − H08291 acid phosphatase 1, soluble ACP1 − NM_014636 Ral guanine nucleotide exchange factor RalGPS1A RALGPS1A − NM_053006 serine/threonine kinase 22B (spermiogenesis associated) STK22B − NM_000220 potassium inwardly-rectifying channel, subfamily J, member 1 KCNJ1 − NM_000633 B-cell CLL/lymphoma 2 BCL2 − NM_003605 O-linked N-acetylgiucosamine (GlcNAc) transferase (UDP-N-acetylglucosamine:polypeptide-N- OGT − acetylglucosaminyl transferase) NM_006114 translocase of outer mitochondrial membrane 40 homolog (yeast) TOMM40 − NM_013404 mesothelin MSLN − NM_020974 signal peptide, CUB domain, EGF-like 2 SCUBE2 − NM_000439 proprotein convertase subtilisin/kexin type 1 PCSK1 − NM_002035 follicular lymphoma variant translocation 1 FVT1 − AL136924 Ras and Rab interactor 2 RIN2 − NM_006020 alkB, alkyfation repair homolog (E. coli) ALKBH − NM_005433 v-yes-1 Yamaguchi sarcoma viral oncogene homolog 1 YES1 − NM_003423 zinc finger protein 43 (HTF6) ZNF43 − AF056490 phosphodiesterase 8A PDE8A − NM_033480 F-box only protein 9 FBXO9 − NM_022789 interleukin 17E IL17E − NM_007150 zinc finger protein 185 (LIM domain) ZNF185 − NM_017450 BAI1-associated protein 2 BAIAP2 − AB037762 myelin expression factor 2 MYEF2 − NM_003263 toll-like receptor 1 TLR1 − NM_001089 ATP-binding cassette, sub-family A (ABC1), member 3 ABCA3 − NM_018240 kin of IRRE like (Drosophila) KIRREL − NM_003827 N-ethylmaleimide-sensitive factor attachment protein, alpha NAPA − NM_003569 syntaxin 7 STX7 − AB046797 KIAA1577 protein KIAA1577 − AV723914 hypothetical protein LOC164729 LOC164729 − NM_015967 protein tyrosine phosphatase, non-receptor type 22 (lymphoid) PTPN22 − AB007892 CDC5 celt division cycle 5-like (S. pombe) CDC5L − NM_022907 hypothetical protein FLJ23053 FLJ23053 − NM_004379 cAMP responsive element binding protein 1 CREB1 − AB023198 KIAA0981 protein KIAA0981 − NM_024958 chromosome 20 open reading frame 98 C20orf98 − NM_001186 BTB and CNC homology 1, basic leucine zipper transcription factor 1 BACH1 − NM_014639 KIAA0372 gene product KIAA0372 − NM_024641 mannosidase, endo-alpha MANEA − AK056671 upstream regulatory element binding protein 1 UREB1 − NM_003618 mitogen-activated protein kinase kinase kinase kinase 3 MAP4K3 − NM_005443 3′-phosphoadenosine 5′-phosphosulfate synthase 1 PAPSS1 * − NM_022781 ring finger protein 38 RNF38 − NM_003874 CD84 antigen (leukocyte antigen) CD84 − NM_000091 collagen, type IV, alpha 3 (Goodpasture antigen) COL4A3 − NM_000160 glucagon receptor GCGR − NM_006019 phosphodiesterase 1A, calmodulin-dependent PDE1A − NM_012080 family with sequence similarity 16, member A, X-linked FAM16AX − 

1. A polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2, 4, or
 6. 2-50. (canceled)
 51. A polypeptide which is (a) a polypeptide comprising the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6, (b) polypeptide which is a fragment of (a), or (c) a polypeptide which has sequence identity to the polypeptides of (a) or (b) of at least 70%.
 52. A method of diagnosing or treating a disease comprising reacting or administering to a subject in need thereof, respectively, a polypeptide of claim
 1. 53. A method of diagnosing or treating a disease comprising administering to a subject in need thereof a polynucleotide which encodes a polypeptide of claim
 1. 54. A method of diagnosing or treating a disease comprising administering to a subject in need thereof, respectively, a recombinant cell which expresses a polypeptide of claim
 1. 55. A method of diagnosing or treating a disease comprising administering to a subject in need thereof an antibody which is directed against a polypeptide of claim
 1. 56. A method according to claim 52 wherein said disease is cancer.
 57. A method according to claim 53 wherein said disease is cancer.
 58. A method according to claim 54 wherein said disease is cancer.
 59. A method according to claim 55 wherein said disease is cancer.
 60. A method according to claim 56 wherein said cancer is lung cancer, breast cancer, prostate cancer, colon cancer, cervix cancer, uterus cancer, larynx cancer, stomach cancer, liver cancer, Ewings sarkoma, acute lymphoid leukemia, chronic myeloid leukemia, apoptosis resistant leukemia, MDR lung cancer, pancreas cancer, gastric cancer, kidney cancer, gliomas, melanomas, chronic lymphoid leukemia, and/or lymphoma.
 61. A method according to claim 57 wherein said cancer is lung cancer, breast cancer, prostate cancer, colon cancer, cervix cancer, uterus cancer, larynx cancer, stomach cancer, liver cancer, Ewings sarkoma, acute lymphoid leukemia, chronic myeloid leukemia, apoptosis resistant leukemia, MDR lung cancer, pancreas cancer, gastric cancer, kidney cancer, gliomas, melanomas, chronic lymphoid leukemia, and/or lymphoma.
 62. A method according to claim 58 wherein said cancer is lung cancer, breast cancer, prostate cancer, colon cancer, cervix cancer, uterus cancer, larynx cancer, stomach cancer, liver cancer, Ewings sarkoma, acute lymphoid leukemia, chronic myeloid leukemia, apoptosis resistant leukemia, MDR lung cancer, pancreas cancer, gastric cancer, kidney cancer, gliomas, melanomas, chronic lymphoid leukemia, and/or lymphoma.
 63. A method according to claim 59 wherein said cancer is lung cancer, breast cancer, prostate cancer, colon cancer, cervix cancer, uterus cancer, larynx cancer, stomach cancer, liver cancer, Ewings sarkoma, acute lymphoid leukemia, chronic myeloid leukemia, apoptosis resistant leukemia, MDR lung cancer, pancreas cancer, gastric cancer, kidney cancer, gliomas, melanomas, chronic lymphoid leukemia, and/or lymphoma.
 64. A pharmaceutical composition or a kit comprising the polypeptide of claim 1 in a pharmaceutically effective amount and a diluent, a carrier and/or an adjuvant.
 65. A method of diagnosing and/or treating cancer comprising administering reacting or to a subject in need thereof, respectively, a pharmaceutical composition of claim
 64. 66. A pharmaceutical composition or a kit comprising at least one of (I), (I) or (II): (I) at least one of the following polypeptides: (a) D-G-E-D-A-A-V (SEQ ID NO:32), (b) (D/Q)-G-(IN)C-R-N-(Q-R)-R-(Q/P) (SEQ ID NO:33), (c) F-A-D-S (SEQ ID NO:34), (d) G-P-D-G-(I/L)-V-A-D (SEQ ID NO:35), (e) P-G-E-V-S-(K/Q)-(I/L) (SEQ ID NO: 36), (f) A-T-Q-A-Y-A-A-V-R-P-I-P-A-S-K (SEQ ID NO:37), (g) D-S-G-L-D-I-A-V-E-Y-S-D-R (SEQ ID NO:38), (h) G-D-V-P-Y-D-L-S-P-E-E-K (SEQ ID NO: 39), (i) SEQ ID NO: 41, 43, 44, 45, or a fragment thereof, wherein the polypeptide or fragment has cytotoxic activity, (II) at least one of the following polynucleotides: (i) a polynucleotide of SEQ ID NO: 40 or 42, a polypeptide encoding portion thereof or a complement thereof, (ii) a polynucleotide corresponding to the sequence of (i) within the scope of degeneracy of the genetic code, or a complement thereof (iii) a polynucleotide comprising a nucleic acid sequence which hybridizes with the sequence of (i) and/or (ii) under stringent hybridizing conditions; (III) an inhibitor of a target substance wherein said target substance comprises at least one member selected from Table 3, Table 4, or Table
 5. 67. A method for the diagnosis or treatment of cancer in a subject, comprising reacting or administering to a subject in need thereof, respectively, a pharmaceutical composition or a kit of claim
 66. 68. A pharmaceutical composition or a kit comprising as an active agent a combination of APIT and at least one inhibitor of a substance of Table 3, Table 4, or Table
 5. 